Patent Application
20250034662
The present application contains a Sequence Listing that has been submitted electronically and is hereby incorporated by reference herein in its entirety. The electronic Sequence Listing is named Sequence Listing_ST26, which was created on Dec. 1, 2022 and is 44,221 bytes in size.
The Psilocybe genus of mushrooms is well known for the synthesis of valuable psychoactive compounds such as psilocybin (as well as psilocin, baeocystin, aeruginascin, etc.). For instance, psilocybin is especially of commercial interest for various reasons. Of note, psilocybin has been classified as a “breakthrough therapy” for depression by the FDA. Chemically, psilocybin is a tryptamine derivative with a structure similar to the mammalian neurotransmitter serotonin. Its pharmacological activity seems to mimic serotonin in the central nervous system, with a high affinity for the 5-HT2A receptor subtype, typical of other hallucinogenic tryptamines.
Psilocybe mushrooms can be grown from Psilocybe spores. The Psilocybe mushrooms may be intended to be edible and thus ingested by humans. Or the Psilocybe mushrooms may be the starting point for further processing to obtain psychoactive compounds such as psilocybin. In any event, there is commercial interest in cultivating Psilocybe mushrooms, or producing psilocybin or psilocybin intermediates in culture, that are safe for ingestion or further processing.
In embodiments, a method of detecting contamination in Psilocybe spores, Psilocybe tissue, or a cultured host organism that expresses Psilocybe genes comprises obtaining a sample including nucleic acids from Psilocybe spores, Psilocybe tissue, or the cultured host organism that expresses Psilocybe genes (hereinafter generically referred to as “Psilocybe sample”), contacting the sample with primers for amplifying target nucleic acid sequences, amplifying any of the target nucleic acid sequences when present among the nucleic acids to obtain amplicons, and detecting the amplicons upon amplification of the target nucleic acid sequence. In embodiments, the primers include primers for amplifying (i) a bacterial target nucleic acid sequence, (ii) a Psilocybe target nucleic acid sequence, and/or (iii) a fungal target nucleic acid sequence.
For instance, the primers may include primers for amplifying a bacterial target nucleic acid sequence and a Psilocybe target nucleic acid sequence. The bacterial target nucleic acid sequence may be any of a 16S target nucleic acid sequence, E. coli target nucleic acid sequence, or Salmonella target nucleic acid sequence. For instance, the bacterial target nucleic acid sequence may include a 16S target nucleic acid sequence. Or the bacterial target nucleic acid sequence may include an E. coli target nucleic acid sequence and/or Salmonella target nucleic acid sequence. The Psilocybe target nucleic acid sequence may be any of a PsiK, PsiM, PsiH, or PsiD target nucleic acid sequence. The Psilocybe target nucleic acid sequence may include a target nucleic acid sequence from each of PsiK and PsiM. The Psilocybe target nucleic acid sequence may include a target nucleic acid sequence from each of PsiK, PsiM, and PsiD.
In embodiments, the Psilocybe sample originates from P. cubensis, P. semilanceata, P. azurescens, P. tampanensis, P. zapotecorum, P. cyanescens, P. caerulescens, P. mexicana, P. caerulipses, P. stuntzii, P. baeocystis, P. bohemica, P. weilii, or P. hoogshagenii. For instance, the Psilocybe sample may originate from P. cubensis.
In embodiments, the Psilocybe genes expressed in the cultured host organism originate from P. cubensis, P. semilanceata, P. azurescens, P. tampanensis, P. zapotecorum, P. cyanescens, P. caerulescens, P. mexicana, P. caerulipses, P. stuntzii, P. baeocystis, P. bohemica, P. weilii, or P. hoogshagenii. For instance, the Psilocybe genes may originate from P. cubensis.
In embodiments, the primers further include primers for amplifying a fungal target nucleic acid sequence. The fungal target nucleic acid sequence may be an Internal Transcribed Spacer (ITS) target nucleic acid sequence.
In embodiments, detection of the amplicons includes quantification of the amplicons. For instance, quantification of amplicons amplified from the bacterial target nucleic acid sequence may be a measure of bacterial contamination. Further, quantification of amplicons amplified from the fungal target nucleic acid sequence may be a measure of fungal contamination. But quantification of amplicons amplified from the Psilocybe target nucleic acid sequence may be a confirmatory measure of presence of Psilocybe spores in the sample.
In embodiments, a polymerase chain reaction (PCR) is performed to amplify the target nucleic acid sequences. For instance, the PCR may be a real-time polymerase chain reaction (qPCR). And multiplex amplification may be performed to amplify the target nucleic acid sequences.
In embodiments, probes are hybridized to the amplicons to detect the amplicons. The probes may include a fluorescent label. And the probes may have different fluorescent labels based on different amplicon sequences to which the probes hybridize. Further, the probes may have a probe structure that includes a fluorophore and a quencher. Example fluorophores include 56-FAM, 56-ROXN, 5TEX615, and 5HEX. Example quenchers include 3IABkFQ, 3IAbRQSp, and ZEN.
In embodiments, the primers include a primer having a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOS: 8-21. In embodiments, the probes include a probe having a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOS: 22-29. Any of the primers and/or probes may hybridize to regions that have less than 1% genomic variation in a sequence alignment performed for multiple Psilocybe strains.
In embodiments, the bacterial and Psilocybe target nucleic acid sequences include a target nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 1-6. In embodiments, the fungal target nucleic acid sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.
In embodiments, a kit for detecting contamination in a Psilocybe sample comprises (i) primers for amplifying a bacterial target nucleic acid sequence, a Psilocybe target nucleic acid sequence, and/or a fungal target nucleic acid sequence, and (ii) probes for detecting the amplicons amplified from any of those target nucleic acid sequences. For instance, the kit may comprise primers for amplifying a bacterial target nucleic acid sequence and a Psilocybe target nucleic acid sequence and probes for detecting amplicons of the bacterial target nucleic acid sequence and the Psilocybe target nucleic acid sequence. The probes may include a fluorescent label.
In embodiments, the kit further comprises reagents including any of a lysis buffer, magnetic beads, a binding buffer, a wash solution, an elution solution, a DNA polymerase, or dNTPs. The kit may also further comprise instructions for performing an assay (or a test) to detect contamination in a Psilocybe sample.
In embodiments, the bacterial target nucleic acid sequence is any of a 16S target nucleic acid sequence, E. coli target nucleic acid sequence, or Salmonella target nucleic acid sequence nucleic acid sequence and the Psilocybe target nucleic acid sequence is any of a PsiK, PsiM, PsiH, or PsiD target nucleic acid sequence. For instance, the bacterial target nucleic acid sequence may include a 16S target nucleic acid sequence. Or the bacterial target nucleic acid sequence may include an E. co/i target nucleic acid sequence and/or Salmonella target nucleic acid sequence. The Psilocybe target nucleic acid sequence may be any combination of PsiK, PsiM, PsiH, or PsiD target nucleic acid sequences. For example, the Psilocybe target nucleic acid sequence may include a target nucleic acid sequence from each of PsiK and PsiM or may include a target nucleic acid sequence from each of PsiK, PsiM, and PsiD.
In embodiments, the kit comprises primers for amplifying a fungal target nucleic acid sequence and a probe for detecting amplicons of the fungal target nucleic acid sequence. The fungal target nucleic acid sequence may be an Internal Transcribed Spacer (ITS) target nucleic acid sequence.
In embodiments, the probes have different fluorescent labels based on different amplicons sequences to which the probes hybridize. The probes may have a probe structure that includes a fluorophore and a quencher.
In embodiments, the primers include a primer having a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOS: 8-21. In embodiments, the probes include a probe having a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOS: 22-29. Any of the primers and/or probes may hybridize to regions that have less than 1% genomic variation in a sequence alignment performed for multiple Psilocybe strains.
In embodiments, the bacterial and Psilocybe target nucleic acid sequences include a target nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 1-6. In embodiments, the fungal target nucleic acid sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.
Those of skill in the art will understand that the figures described below are for illustrative purposes only. The figures are not intended to be limiting in any way.
There is commercial interest in cultivating Psilocybe mushrooms, or producing psilocybin or psilocybin intermediates from a cultured host organism that expresses Psilocybe genes, that are safe for ingestions or further processing. Bacterial contamination and/or non-Psilocybe fungal contamination can negatively affect the growth, productivity, yield, and/or safety of Psilocybe mushrooms or the cultured host organism. Antibiotics can often be added to reduce bacterial contamination, but not all fungi or potential host organisms tolerate antibiotics and not all users of psilocybin or Psilocybe mushrooms tolerate residual antibiotics.
As a result, there exists a desire to screen Psilocybe tissue or a cultured host organism expressing Psilocybe genes for bacterial contamination and/or non-target fungal contamination. And there exists a desire to screen Psilocybe spores for bacterial contamination and/or non-target fungal contamination prior to inoculation. This is particularly important in the growth of edible mushrooms like Psilocybe where it is preferred to grow these mushrooms in the absence of antibiotics to avoid antibiotic contamination of edible mushrooms. The spawning stage of mycelium growth is particularly sensitive to bacterial contamination as the process can take 2-4 weeks for fully mature mycelium to grow. Injecting bacterially contaminated spores into spawning grains or medium will result in contaminated growth and failed Psilocybe growth. Further, assaying/testing Psilocybe spores may prevent misidentification of spores from poisonous mushrooms like Galarina marginata or Cortinarius as being Psilocybe spores.
In embodiments, a method of detecting contamination in a Psilocybe sample thus comprises obtaining a sample including nucleic acids from Psilocybe spores, Psilocybe tissue, or a cultured host organism that expresses Psilocybe genes, contacting the sample with primers for amplifying target nucleic acid sequences, amplifying any of the target nucleic acid sequences when present among the nucleic acids to obtain amplicons, and detecting the amplicons upon amplification of the target nucleic acid sequence. In embodiments, the primers include primers for amplifying (i) a bacterial target nucleic acid sequence, (ii) a Psilocybe target nucleic acid sequence, and/or (iii) a fungal target nucleic acid sequence.
The method may be used for assaying (i.e., testing) a Psilocybe sample for contamination from bacteria and other fungal (e.g., yeast and mold) sources. In the case of Psilocybe spores, the contamination may be quantified (i.e., measured) so as to determine whether the Psilocybe spores are suitable for growing Psilocybe mushrooms. In other words, the method may be used to screen Psilocybe spores, Psilocybe tissue, or a cultured host organism that expresses Psilocybe genes for contamination. Contaminated samples may then be discarded in favor of samples with less contamination (e.g., without significant and/or detectable contamination). Embodiments are described in greater detail below.
In embodiments, the Psilocybe sample is subject to assaying/testing for contamination. The Psilocybe spores or tissue may originate from P. cubensis, P. semilanceata, P. azurescens, P. tampanensis, P. zapotecorum, P. cyanescens, P. caerulescens, P. mexicana, P. caerulipses, P. stuntzii, P. baeocystis, P. bohemica, P. weilii, or P. hoogshagenii. Likewise, the Psilocybe genes expressed in the cultured host organism may originate from P. cubensis, P. semilanceata, P. azurescens, P. tampanensis, P. zapotecorum, P. cyanescens, P. caerulescens, P. mexicana, P. caerulipses, P. stuntzii, P. baeocystis, P. bohemica, P. weilii, or P. hoogshagenii. For instance, the Psilocybe sample may originate from P. cubensis. In embodiments, the Psilocybe spores, tissue, or genes originate from P. cubensis.
Psilocybe spores may be obtained from commercial sources (e.g., sporeworks.com, premiumspores.com, mushroom.com, inoculatetheworld.com, etc.) or may be otherwise prepared. Spores may be provided in a syringe (e.g., containing an aqueous suspension/solution of the spores). To assay/test for contamination, a sample is obtained of the Psilocybe spores (e.g., a sample of an aqueous suspension/solution of the spores).
Psilocybe tissue may be obtained from any part of the Psilocybe fungus, such as the mycelium or the fruiting body (which includes the trama, the hymenium, and the pileipellis). The tissue can be fresh (hydrated) or dried. The tissue could be an intact mushroom, one or more tissue layers of the mushroom (in combination or isolated), or a cellular sample. To assay/test for contamination, a sample is obtained of the Psilocybe tissue (e.g., a sample of an aqueous suspension/solution of the cells or homogenized tissue).
A host organism that expresses Psilocybe genes can be used to produce psilocybin or psilocybin intermediates in culture. This has the potential to be a more environmentally benign process with higher throughput.
For example, engineered psilocybin biosynthesis genes can be expressed in a recombinant host organism. The biosynthetic production of psilocybin starts with L-tryptophan, which is converted into tryptamine by tryptophan decarboxylase. Tryptamine is next converted into 4-hydroxytryptamine by a cytochrome P450 containing monooxygenase (PcPsiH). Cytochrome P450 enzymes are characterized by their dependency on a cytochrome P450 reductase (CPR), which facilitates electron transfer between NADPH and cytochrome P450 enzymes. 4-hydroxytryptamine is next converted into norbaeocystin by a 4-hydroxytryptamine kinase encoded by PcPsiK, which facilitates the 4-O-phosphorylation reaction. Finally, an N-methyltransferase encoded by PcPsiM mediates the iterative methyl transfer of norbaeocystin to baeocystin then to psilocybin. The recombinant host can be modified to include genes encoding the enzymes involved in the psilocybin biosynthesis pathway.
Alternatively, endogenous pathways of the recombinant host can be modified to produce high purity psilocybin and/or intermediates of psilocybin. For example, production of psilocybin can be increased by promoting expression of CPR in the host organism.
Recombinant host organisms include, for example, E. coli, Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica. Genes that can be inserted or engineered into the recombinant host include PsiK, PsiM, PsiH, and PsiD, and genes encoding other enzymes involved in the psilocybin biosynthesis pathway. The engineered enzymes act on substrates in the psilocybin biosynthetic pathway (e.g., L-tryptophan and/or 4-hydroxy-L-tryptophan) to produce intermediates of psilocybin and psilocybin itself. See, e.g., Gibbons Jr., Bioengineered 12:8863 (2021); Milne, Metabolic Engineering 60:25 (2020); U.S. 2021/0147888 (the disclosures of which are incorporated herein).
In embodiments, a sample including nucleic acids is obtained from the Psilocybe sample. The nucleic acids may be DNA and/or RNA. In that respect, nucleic acids are polymers of nucleotides (e.g., ribonucleotides and deoxyribonucleotides, both natural and non-natural, including adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)) such polymers being DNA, RNA, and their subcategories, such as cDNA, mRNA, etc. A nucleic acid may be single-stranded or double-stranded and will generally contain 5′-3′ phosphodiester bonds, although in some cases, nucleotide analogs may have other linkages.
Nucleic acids will typically include naturally occurring bases (adenine (A), cytosine (C), guanine (G), thymine (T), and/or uracil (U)) but it is possible that they contain non-natural bases in embodiments. (The example of non-natural bases include those described in, e.g., Seela et al. (1999) Helv. Chim. Acta 82:1640. Certain bases used in nucleotide analogs act as melting temperature (Tm) modifiers. For example, some of these include 7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.), pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC, etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303. Other representative heterocyclic bases include, e.g., hypoxanthine, inosine, xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 6-azacytidine; 5-fluorocytidine; 5-chlorocytidine; 5-iodocytidine; 5-bromocytidine; 5-methylcytidine; 5-propynylcytidine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil; 5-ethynyluracil; 5-propynyluracil, and the like.)
In embodiments, the nucleic acids include genomic DNA. In other embodiments, the nucleic acids will include mRNA or cDNA produced from the mRNA for purposes of detecting gene expression as opposed to genomic DNA per se. As is well known, the genetic framework of an organism is encoded in the double-stranded sequence of nucleotide bases in the DNA which is contained in the cells of the organism. The genetic content of a particular segment of DNA, or gene, is manifested only upon production of the gene product (RNA or protein) encoded by the gene. To produce a gene product, a complementary copy of one strand of the DNA double helix (the “coding” strand) is produced by polymerase enzymes, resulting in a specific sequence of RNA. When this particular type of RNA contains the genetic message from the DNA for production of a protein, it is called messenger RNA (mRNA).
A common approach to the study of gene expression is the production of complementary DNA (cDNA). In this technique, the RNA (e.g., mRNA) molecules from an organism are isolated from an extract of the cells (or tissues) of the organism. From these purified mRNA molecules, cDNA copies may be made using the enzyme reverse transcriptase (RT) or DNA polymerases having RT activity, which results in the production of single-stranded cDNA molecules.
Reverse transcriptases are a class of polymerases characterized as RNA dependent DNA polymerases. Known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA dependent DNA polymerase (Verma, Biochem. Biophys. Acta 473:1(1977)). The enzyme has 5′-3′ RNA directed DNA polymerase activity, 5′-3′ DNA directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5′ and 3′ ribonuclease specific for the RNA strand for RNA DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3′-5′ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity has been presented by Berger et al., Biochemistry 22:2365 2372 (1983). Another reverse transcriptase which is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271 279 (1986) and Kotewicz, M. L., et al., Gene 35:249 258 (1985). M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No. 5,244,797.
Conventional protocols for obtaining DNA or RNA from cells are described in the literature. See, e.g., Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York (1993). Conventional DNA isolation protocols generally entail suspending the cells in a solution and using enzymes and/or chemicals to lyse the cells, thereby releasing the DNA contained within the cells into the resulting lysate solution. The enzymes and/or chemicals may be provided in a lysis buffer. For isolation of RNA, the conventional lysis and solubilization procedures include measures for inhibition of ribonucleases and contaminants to be separated from the RNA including DNA.
Any one of a number of different known methods for lysing or disrupting cells to release nucleic acid materials contained therein are suitable for use in producing a medium from cells. For example, in order to cause a cell with a relatively hard cell wall, such as a fungus cell, to release the nucleic acid material contained therein one may need to use harsher treatments such as potent proteases and mechanical shearing with a homogenizer or disruption with sound waves using a sonicator. Once the nucleic acid material is released from cells lysed or disrupted, cellular debris can be removed using a number of different known techniques or combination of techniques. The solution of lysed or disrupted cells may be centrifuged to remove particulate cell debris. The supernatant may then be further processed by adding a second solution to the supernatant which causes a precipitate of additional other material to form, and then removing the precipitate from the resulting solution by centrifugation.
Many conventional protocols generally entail use of phenol or an organic solvent mixture containing phenol and chloroform to extract additional cellular material such as proteins and lipids from a conventional lysate solution produced as described above. The phenol/chloroform extraction step is generally followed by precipitation of the nucleic acid material remaining in the extracted aqueous phase by adding ethanol to that aqueous phase. The precipitate is typically removed from the solution by centrifugation, and the resulting pellet of precipitate is allowed to dry before being resuspended in water or a buffer solution for further processing or analysis.
Glass particles, silica particles, silica gel, and mixtures thereof have been configured in various different forms to produce matrices capable of reversibly binding nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents. Such matrices are designed to remain bound to the nucleic acid material while the matrix is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components. The nucleic acid material is then eluted from the matrix by exposing the matrix to an elution solution, such as water or an elution buffer. Numerous commercial sources offer silica-based matrices designed for use in centrifugation and/or filtration isolation systems. See, e.g., Wizard™ DNA purification systems line of products from Promega Corporation (Madison, Wisconsin, USA); or the QiaPrep™ line of DNA isolation systems from Qiagen Corp. (Chatsworth, California, USA).
Magnetically responsive particles (“magnetic particles”) and methods for using magnetic particles have been developed for the isolation of nucleic acid materials. Several different types of magnetic particles designed for use in nucleic acid isolation are described in the literature, and many of those types of particles are available from commercial sources. Such magnetic particles generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and those designed to do so through at least one intermediary label.
The magnetic particles designed to bind nucleic acid materials indirectly are generally used to isolate a specific nucleic acid material, such as mRNA, according to the following basic isolation procedure. First, a medium containing a nucleic acid material is placed in contact with a label capable of binding to the nucleic acid material of interest. For example, one such commonly employed label, biotinylated oligonucleotide deoxythymidine (oligo-dT), forms hydrogen bonds with the poly-adenosine tails of mRNA molecules in a medium. Each label so employed is designed to bind with a magnetically responsive particle, when placed into contact with the particle under the proper binding conditions. For example, the biotin end of a biotinylated oligo-dT/mRNA complex is capable of binding to streptavidin moieties on the surface of a streptavidin coated magnetically responsive particle. Several different commercial sources are available for streptavidin magnetic particles and reagents designed to be used in mRNA isolation using biotinylated oligo-dT as described above. See, e.g., PolyATtract™ Series9600™ mRNA Isolation System from Promega Corporation; or the ProActive™ line of streptavidin coated microsphere particles from Bangs Laboratories (Carmel, Indiana, USA).
Magnetic particles and label systems have also been developed which are capable of indirectly binding and isolating other types of nucleic acids, such as double-stranded and single-stranded PCR templates. See, e.g., BioMag™ superparamagnetic particles from Advanced Magnetics, Inc. (Cambridge, Massachusetts, USA). Indirect binding magnetic separation systems for nucleic acid isolation or separation all require at least three components, i.e. magnetic particles, a label, and a medium containing the nucleic acid material of interest. The label/nucleic acid binding reaction and label/particle binding reaction often require different solution and/or temperature reaction conditions from one another.
Other types of magnetic particles have also been developed for use in the direct binding and isolation of biological materials, particularly nucleic acids. One such particle type is a magnetically responsive glass bead that may be of a controlled pore size. See, e.g., Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park, New Jersey, USA); or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169; or 4,297,337. Another type of magnetically responsive particles designed for use in direct binding and isolation of biological materials, particularly nucleic acids, are particles comprised of agarose embedded with smaller ferromagnetic particles and coated with glass. See, e.g., U.S. Pat. No. 5,395,498.
The external magnetic field may be suitably generated using any one of a number of different known means. For example, one can position a magnet on the outer surface of a container of a solution containing the particles, causing the particles to migrate through the solution and collect on the inner surface of the container adjacent to the magnet. The magnet can then be held in position on the outer surface of the container such that the particles are held in the container by the magnetic field generated by the magnet, while the solution is decanted out of the container and discarded. A second solution can then be added to the container, and the magnet removed so that the particles migrate into the second solution. Alternatively, a magnetizable probe could be inserted into the solution and the probe magnetized, such that the particles deposit on the end of the probe immersed in the solution. The probe could then be removed from the solution, while remaining magnetized, immersed into a second solution, and the magnetic field discontinued permitting the particles go into the second solution. Commercial sources exist for magnets designed to be used in both types of magnetic removal and transfer techniques described in general terms above. See, e.g., MagneSphere™ Technology Magnetic Separation Stand or the PolyATract™ Series 9600™ Multi-Magnet, both available from Promega Corporation; Magnetight Separation Stand (Novagen, Madison, Wisconsin, USA); or Dynal Magnetic Particle Concentrator (Dynal, Oslo, Norway).
The complex of nucleic acids with the particles that is removed from the medium may be washed at least once by being rinsed in a wash solution. The wash solution used in this additional step of the method may comprise a solution capable of removing contaminants from the silica magnetic particle. The wash solution may comprise a salt and a solvent, such as an alcohol. The concentration of alcohol in embodiments of the wash solution may be at least 30% by volume, at least 40% by volume, or at least 50% by volume. The alcohol so used may be ethanol or isopropanol. The salt may be in the form of a buffer, such as in the form of an acetate buffer. The concentration of salt in the wash solution is sufficiently high to ensure the nucleic acid material is not eluted from the silica magnetic particles during the wash step(s).
The nucleic acid material may then be eluted from the particles by exposing the complex to an elution solution. The elution solution may be an aqueous solution of low ionic strength, such as water or a low ionic strength buffer at about a pH at which the nucleic acid material is stable and substantially intact. Any aqueous solution with an ionic strength at or lower than TE buffer (i.e. 10 mM Tris-HCl, 1 mM ethylenediamine-tetraacetic acid (EDTA), pH 8.0) is suitable for use in the elution steps. The elution solution may be buffered to a pH between about 6.5 and 8.5 or buffered to a pH between about 7.0 and 8.0. TE Buffer and distilled or deionized water are exemplary elution solutions for use. The low ionic strength of such an elution solution ensures the nucleic acid material is released from the particle. Other elution solutions suitable for use in the methods will be readily apparent to one skilled in this art.
In embodiments, the method includes contacting the sample with primers for amplifying target nucleic acid sequences, amplifying any of the target nucleic acid sequences when present among the nucleic acids to obtain amplicons, and detecting the amplicons upon amplification of the target nucleic acid sequences. In that respect, bacterial contamination and/or fungal (e.g., yeast and mold) contamination may be quantified (i.e., measured) with respect to the sample.
Amplification refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Such nucleic acid amplification reactions include non-isothermal amplification methods such as polymerase chain reaction (PCR), particularly quantitative/real-time PCR or isothermal amplification methods such as NASBA (nucleic acids sequence based amplification), TMA (Transcription mediated amplification), 3SR (self-sustained sequence amplification), SDA (Strand displacement amplification), HDA (helicase dependent amplification, with heat-labile or heat-stabile enzymes), RPA (recombinase polymerase amplification), LAMP (Loop-mediated amplification); or SMAP (SMart Amplification Process). These technologies make use of enzymes, proteins, primers and accessory molecules that are well known to those skilled in the art. The polymerases include any of a Thermus thermophilus (Tth) DNA polymerase, Thermus acquaticus (Taq) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus kodakaraensis KOD DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Sulfolobus solfataricus Dpo4 DNA polymerase, Thermus pacificus (Tpac) DNA polymerase, Thermus eggertsonii (Teg) DNA polymerase and Thermus flavus (Tfl) DNA polymerase and the polymerases of phages, e.g., Phi29-phage, Phi29 like phages such as Cp-1, PRD-1, Phi 15, Phi 21, PZE, PZA, Nf, M2Y, B103, SF5, GA-1, Cp-5, Cp-7, PR4, PR5, PR722, or L 17. The polymerases also include polymerases from other organisms such as from E. coli, T4, or T7. Other additional proteins may improve the methods, for example helicases, single-stranded binding proteins, other DNA-binding proteins, and recombinases.
In embodiments, PCR is used to perform amplification of the target nucleic acid sequences when present among the nucleic acids to obtain amplicons. PCR is a method that allows exponential amplification of target DNA sequences within a longer double stranded DNA molecule. PCR entails the use of a pair of primers that are complementary to a defined sequence on each of the two strands of the DNA. These primers are extended by a DNA polymerase so that a copy is made of the designated sequence. After making this copy, the same primers can be used again, not only to make another copy of the input DNA strand but also of the short copy made in the first round of synthesis. This leads to logarithmic amplification.
A primer is an oligonucleotide capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., either in the presence of four different nucleoside triphosphates and an agent for extension (e.g., a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer may be single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but may range from 6 to 50 nucleotides, such as from 15-35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template.
A forward primer is a primer that is capable of hybridizing to a region of DNA along the 5′ (coding) strand of DNA. A reverse primer is a primer that is capable of hybridizing to a region of DNA along the 3′ (non-coding) strand of DNA. A primer set or primer pair is a specific combination of at least one forward primer and at least one reverse primer. A primer is specific for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily only to the target nucleic acid. Typically, a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in most cases.
Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the specific amplification of those target sequences which contain the target primer binding sites. The use of sequence-specific amplification conditions enables the specific amplification of those target sequences which contain the complementary primer binding sites. Complementary refers to instances where a nucleic acid molecule can form hydrogen bond(s) with another nucleic acid molecule by either traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules may be less than fully complementary and is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. “Perfectly” or “fully” complementary nucleic acid molecules means those in which all the contiguous residues of a first nucleic acid molecule will hydrogen bond with the same number of contiguous residues in a second nucleic acid molecule, wherein the nucleic acid molecules either both have the same number of nucleotides (i.e., have the same length) or the two molecules have different lengths.
Hybridization refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which only fully complementary nucleic acid strands will hybridize are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art. See, e.g., Sambrook et al., (1989) Molecular Cloning—A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); and Wetmur (1991) Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259.
An amplicon is the DNA sequence generated by a PCR (e.g., qPCR) reaction. In that respect, an amplicon is a PCR product. A target nucleic acid sequence (or “target, “target sequence”, “target region”, or “target nucleic acid”) refers to a region or subsequence of a nucleic acid which is to be amplified and detected. A reaction mixture is a solution containing reagents necessary to carry out a given reaction. And an amplification reaction mixture, which is a solution containing reagents necessary to carry out an amplification reaction, typically contains oligonucleotide primers and a DNA polymerase or ligase in a suitable buffer. A PCR reaction mixture typically contains oligonucleotide primers, a DNA polymerase (most typically a thermostable DNA polymerase), dNTPs, and a divalent metal cation in a suitable buffer. dNTPs are deoxyribose nucleotide triphosphates. They are the building blocks added during a PCR in order to synthesize a new strand. Examples are deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP) (and/or deoxyuridine triphosphate (dUTP)).
A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components.
Real-time PCR, also called quantitative real time PCR, quantitative PCR (Q-PCR/qPCR), or kinetic polymerase chain reaction, is a type of PCR that is used to amplify and simultaneously quantify a targeted DNA molecule. qPCR enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. The procedure follows the general principle of PCR and its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle.
In qPCR, DNA dyes or probes (e.g., fluorescent probes) can be added to the PCR mixture before amplification and used to analyze PCR products (amplicons) during amplification. Sample analysis may occur concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions (as there is no need to remove the samples from their closed containers for further analysis).
The formation of PCR products is monitored in each cycle of the qPCR. The amplification may be measured in thermocyclers which have additional devices for measuring fluorescence signals during the amplification reaction. Several types of real time detection thermocyclers are known in the art.
An example of an instrument capable of performing multiplex real time PCR is the LightCycler (Roche Diagnostics GmbH, Cat. No. 3 531 414 201). It is a fast PCR system enabling kinetic on-line PCR quantification and subsequent analysis of PCR-product melting curves. The optical system of the LightCycler version 2.0 contains one light source, a blue light emitting diode (470 nm LED) and six detection channels. A defined signal threshold is determined for all reactions to be analyzed and the number of cycles Cp required to reach this threshold value is determined for the target nucleic acid as well as for the reference nucleic acids such as the standard or housekeeping gene. The absolute or relative copy numbers of the target molecule can be determined on the basis of the Cp values obtained for the target nucleic acid and the reference nucleic acid. The fluorescence emitted by a sample is separated by a set of dichroic mirrors and filters into different wavelengths that can be recorded in one of the six detection channels. Due to the fluorescent compounds (which are commercially available), this allows detection of the double-stranded DNA-binding dye SYBR Green I, dual color detection with the TAQMAN probe format and 4-color detection with the hybridization probe (Hybprobe) format. Details of the LIGHTCYCLER system are disclosed in WO 97/46707, WO 97/46712 and WO 97/46714.
Similar to the LightCycler system, the Corbett Rotor-Gene Real time PCR Thermocycler (www.corbettresearch.com) is a 4 channel multiplexing system comprising 4 different LEDs as excitation sources and corresponding photodiodes as fluorescent detection units. Another real time PCR instrument is the Biorad iQ Multi-color Real time PCR detection system (Cat. No: 170-8740), which allows for a fluorophore excitation and emission from 400 nm to 700 nm. The system is based on a conventional multiwell heating block for thermocycling, a tungsten lamp as an excitation source, a filter wheel for providing appropriate excitation wavelengths, a second filter wheel for selecting appropriate emission wavelengths and a CCD camera as a detection unit. The instrument has successfully been used in a multiplex assay for the detection of 4 different amplicons generated from targets with more or less equimolar concentrations, using four differently labeled TaqMan probes in the same reaction vessel (Pedersen, S., Bioradiations 107 (2001) 10-11).
In general, there exist different formats for real time detection of amplified DNA, of which the following are well known and commonly used in the art.
DNA Binding Dye Formats: Since the amount of double-stranded amplification product usually exceeds the amount of nucleic acid originally present in the sample to be analyzed, double-stranded DNA specific dyes may be used, which upon excitation with an appropriate wavelength show enhanced fluorescence only if they are bound to double-stranded DNA. Preferably, only those dyes may be used which, like SybrGreenI, for example, do not affect the efficiency of the PCR reaction. Other formats known in the art require the design of a fluorescent labeled hybridization probe (which may only emit fluorescence upon binding to its target nucleic acid).
TaqMan Probe: A single-stranded hybridization probe is labeled with two components. When the first component is excited with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is degraded by the 5′-3′ exonuclease activity of the Taq Polymerase during the subsequent elongation phase. As a result, the excited fluorescent component and the quencher are spatially separated from one another and thus a fluorescence emission of the first component can be measured. See, e.g., U.S. Pat. No. 5,538,848.
Molecular Beacons: These hybridization probes are also labeled with a first fluorescent component and with a quencher such that the labels may be located at both ends of the probe. As a result of the secondary structure of the probe, both components are in spatial vicinity in solution. After hybridization to the target nucleic acids, both components are separated from one another such that the fluorescence emission of the first component can be measured after excitation with light of a suitable wavelength. See, e.g., U.S. Pat. No. 5,118,801.
Single Label Probe (SLP) Format: This detection format includes a single oligonucleotide labeled with a single fluorescent dye at either the 5′- or 3′-end (WO 02/14555). Two different designs can be used for oligo labeling: G-Quenching Probes and Nitroindole-Dequenching probes. In the G-Quenching embodiment, the fluorescent dye is attached to a C at the 5′- or 3′-end of the oligonucleotide. Fluorescence decreases significantly when the probe is hybridized to the target, in case two G's are located on the target strand opposite to C and in position 1 aside of complementary oligonucleotide probe. In the Nitroindole-Dequenching embodiment, the fluorescent dye is attached to Nitroindole at the 5′- or 3′-end of the oligonucleotide. Nitroindole decreases the fluorescent signaling of the free probe. Fluorescence increases when the probe is hybridized to the target DNA due to a dequenching effect.
FRET Hybridization Probes: The FRET Hybridization Probe test format is known to be useful for all kinds of homogenous hybridization assays (Matthews, J. A., and Kricka, L. J., Analytical Biochemistry 169 (1988) 1-25. It is characterized by a pair of two single-stranded hybridization probes which are used simultaneously and are complementary to adjacent sites of the same strand of the amplified target nucleic acid. Both probes are labeled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that a fluorescence emission of the second component can be measured when both hybridization probes bind to adjacent positions of the target molecule to be detected.
When annealed to the target sequence, the hybridization probes must sit very close to each other in a head to tail arrangement. The gap between the labeled 3′ end of the first probe and the labeled 5′ end or the second probe is typically as small as possible, i.e. 1-5 bases. This allows for a close vicinity of the FRET donor compound and the FRET acceptor compound, which is typically 10-100 Angstroms.
As an alternative to monitoring the increase in fluorescence of the FRET acceptor component, it is also possible to monitor fluorescence decrease of the FRET donor component as a quantitative measurement of hybridization events.
In embodiments, the FRET Hybridization Probe format may be used in real time PCR in order to detect the amplified target DNA. Besides PCR and real time PCR, FRET hybridization probes are used for melting curve analysis. In such an assay, the target nucleic acid is amplified first in a typical PCR reaction with suitable amplification primers. The hybridization probes may already be present during the amplification reaction or added subsequently. After completion of the PCR reaction, the temperature of the sample is constitutively increased, and fluorescence is detected as long as the hybridization probe was bound to the target DNA. At melting temperature, the hybridization probes are released from their target, and the fluorescent signal decreases to the background level. This decrease is monitored with an appropriate fluorescence versus temperature-time plot such that a first derivative value can be determined at which the maximum of fluorescence decrease is observed.
All probe based detection formats can be “multiplexed”. More precisely, in one reaction vessel, multiple targets may become amplified with multiple pairs of amplification primers and detected with multiple hybridization probes. In embodiments, the multiple probes are labeled with different detectable fluorescent components in order to detect and discriminate the multiple targets which may be found in the sample. For multiplex detection with the FRET hybridization probe format, it is possible that fluorescein or fluorescein derivatives are used as a FRET donor moiety in combination with different FRET acceptor moieties such as Cy-5, LC-Red-640, or LC-Red 705.
The target nucleic acid sequences include any sequence of a (i) bacterial (e.g., 16S, E. coli, and/or Salmonella) nucleic acid, (ii) Psilocybe (e.g., PsiK, PsiM, PsiH, and/or PsiD) nucleic acid, and/or (iii) fungal (e.g., Internal Transcribed Spacer) nucleic acid.
Exemplary target nucleic acids are described in more detail below.
Of note, sequence identities are discussed herein with respect to nucleic acid sequences (e.g., target nucleic acid sequences, primers, probes, etc.). Sequence identities can be calculated using various publicly available software tools developed by NCBI (Bethesda, Maryland, USA) that can be obtained through the NCBI internet site. Exemplary tools include the BLAST software, also available at the NCBI internet site (www.ncbi.nlm.nih.gov). Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the nucleic acid sequences are also contemplated herein.
It is known that certain gene sequence fragments within most bacteria are essentially identical. These sequence fragments are called “conserved” regions. Because of these conserved regions, it is possible to use consensus primers, which bind to these regions, to amplify nucleic acids of bacteria as a class of microorganisms. The conserved bacterial regions include the 16S region, 23S region, and 5S region.
An exemplary conserved 16S target nucleic acid sequence has the following sequence:
In embodiments, other conserved 16S target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
More specific bacterial target nucleic acid sequences may be amplified and detected. For instance, the bacterial target nucleic acid sequence may be an Escherichia coli or Salmonella target nucleic acid sequence.
Detection of the microorganism E. co/i has been considered as an indicator of the possible presence of enteric pathogens. Indeed, certain E. co/i strains are pathogenic themselves.
An exemplary E. coli target nucleic acid sequence has the following sequence:
In embodiments, other E. coli target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
Salmonella is a genus of bacteria that are causative of severe infections, notably of food or beverage toxi-infections, leading to bacterial enteric illness in both humans and animals, more particularly to salmonellosis, which include gastroenteritis, as well as typhoid and paratyphoid fevers.
An exemplary Salmonella target nucleic acid sequence has the following sequence:
In embodiments, other Salmonella target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
Psilocybe target nucleic acids include a sequence from any of a PsiK, PsiM, PsiH, or PsiD nucleic acid. In embodiments, the target nucleic acid sequences include sequences from any combination of PsiK, PsiM, PsiH, or PsiD nucleic acids. For instance, the Psilocybe target nucleic acid sequence may include a target nucleic acid sequence from each of PsiK and PsiM or may include a target nucleic acid sequence from each of PsiK, PsiM, and PsiD. Further, the target nucleic acid sequence may include a target nucleic acid sequence from each of PsiK, PsiM, PsiH, and PsiD.
PsiK: A PsiK nucleic acid (gene) encodes a Psilocybe PsiK enzyme, which is a kinase (e.g., 4-hydroxytryptamine kinase). The PsiK kinase can catalyze the phosphorylation of the phenolic oxygen of 4-hydroxytryptamine to norbaeocystin. The PsiK kinase can also catalyze the phosphorylation of psilocin to psilocybin.
An exemplary PsiK target nucleic acid sequence has the following sequence:
In embodiments, other PsiK target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
PsiM: A PsiM nucleic acid (gene) encodes a Psilocybe PsiM enzyme, which is a methyl transferase (e.g., psilocybin synthase). The PsiM methyl transferase can catalyze the alkylation of the primary amine in norbaeocystin to baecystin. The PsiM methyl transferase can catalyze another alkylation when the secondary amine of baecystin becomes a tertiary amine of psilocybin.
An exemplary PsiM target nucleic acid sequence has the following sequence:
In embodiments, other PsiM target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
PsiH: A PsiH nucleic acid (gene) encodes a Psilocybe PsiH enzyme, which is a monooxygenase (e.g., tryptamine 4-monooxygenase). The PsiH monooxygenase can catalyze the oxidative hydroxylation of the phenyl ring of tryptamine to 4-hydroxytryptamine. PsiD: A PsiD nucleic acid (gene) encodes a Psilocybe PsiD enzyme, which is a decarboxylase (e.g., L-tryptophan decarboxylase). The PsiD decarboxylase can catalyze the decarboxylation of an aliphatic carboxylic acid (i.e., release carbon dioxide) so as to catalyze L-tryptophan to tryptamine and 4-hydroxy-L-tryptophan to 4-hydroxytryptamine.
An exemplary PsiD target nucleic acid sequence has the following sequence:
In embodiments, other PsiD target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
It is known that certain gene sequence fragments within most fungus (e.g., yeast and mold) are essentially identical. These sequence fragments are termed “conserved” regions. Because of these conserved regions, it is possible to use consensus primers, which bind to these regions, to amplify nucleic acids of fungi as a class of microorganisms (e.g., total yeast and mold).
The fungal target nucleic acid sequences include sequences of the mitochondrial small subunit ribosomal RNA genes (SSU rDNA) or the Internal Transcribed Spacer (ITS) sequences of the nuclear ribosomal RNA gene regions. Fungal rRNA genes are organized in units, each of which encodes three mature subunits of 18S (small subunit), 5.8S, and 28S (large subunit). These subunits are separated by two Internal Transcribed Spacers, ITS1 and ITS2. In addition, the transcriptional units are separated by non-transcribed spacer sequences (NTSs).
An exemplary fungal ITS target nucleic acid sequence has the following sequence:
In embodiments, other fungal ITS target nucleic acid sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this sequence.
Primers for amplifying one or more target nucleic acid sequences (i.e., primers specific for the target nucleic acid sequences) include primers that hybridize with primer-binding sequences found within any of a (i) bacterial (e.g., 16S, E. co/i, and/or Salmonella) target nucleic acid sequence, (ii) Psilocybe (e.g., PsiK, PsiM, PsiH, and/or PsiD) target nucleic acid sequence, and/or (iii) fungal (e.g., Internal Transcribed Spacer) target nucleic acid sequence.
A primer refers to oligonucleotides that hybridize in a sequence specific manner to a complementary nucleic acid molecule (e.g., a nucleic acid molecule comprising a target sequence). In some embodiments, a primer will comprise a region of nucleotide sequence that hybridizes to at least 6, at least 8, at least 10, at least 15, at least 20, at least 25 nucleotides (e.g., 10 to 60 nucleotides) of a target nucleic acid (i.e., will hybridize to a sequence of the target nucleic acid). In general, a primer sequence is identified as being either “complementary” (i.e., complementary to the coding or sense strand (+)), or “reverse complementary” (i.e., complementary to the anti-sense strand (−)). In embodiments, the primer is an oligonucleotide that acts as a point of initiation of a template-directed synthesis using methods such as PCR under appropriate conditions (e.g., in the presence of four different nucleotide triphosphates and a polymerization agent, such as DNA polymerase in an appropriate buffer solution containing any necessary reagents and at suitable temperature(s)). Such a template directed synthesis is also called “primer extension.” For example, a primer pair may be designed to amplify a region of DNA using PCR. Such a pair will include a “forward primer” and a “reverse primer” that hybridize to complementary strands of a DNA molecule and that delimit a region to be synthesized and/or amplified.
Probes for detecting one or more amplicons obtained from amplifying target nucleic acid sequences (i.e., probes specific for the target nucleic acid sequences) include probes that hybridize with probe-binding sequences within amplicons obtained from amplifying a target nucleic acid sequence found within a (i) bacterial (e.g., 16S, E. co/i, and/or Salmonella) target nucleic acid sequence, (ii) Psilocybe (e.g., PsiK, PsiM, PsiH, and/or PsiD) target nucleic acid sequence, and/or (iii) fungal (e.g., Internal Transcribed Spacer) target nucleic acid sequence.
The probe may be labeled with a first fluorescent component/moiety/label. This hybridization probe may be any kind of hybridization probe which is used in real time PCR such as TAQMAN probe, a molecular beacon, or a single labeled probe. In other embodiments, the probe comprises at least a second hybridization probe labeled with a second fluorescent component/moiety/label.
Examples of particular fluorophores that can be used in the probes are known to those of skill in the art (see, e.g., U.S. Pat. No. 5,866,366 to Nazarenko et al.). Exemplary fluorophores include 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC), -6-carboxy-fluorescein (HEX), and TET (Tetramethyl fluorescein); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosanilin; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); sulforhodamine B; sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); riboflavin; rosolic acid and terbium chelate derivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein; boron dipyrromethene difluoride (BODIPY); acridine; stilbene; 6-carboxy-X-rhodamine (ROX); Texas Red; Cy3; Cy5, VIC® (Applied Biosystems); LC Red 640; LC Red 705; and Yakima yellow among others.
Other suitable fluorophores include, for example, those available from Molecular Probes (Eugene, Oregon, USA). In embodiments, a fluorophore is used as a donor fluorophore and/or as an acceptor fluorophore.
Acceptor fluorophores are fluorophores which absorb energy from a donor fluorophore, for example in the range of about 400 to 900 nm (such as in the range of about 500 to 800 nm). Acceptor fluorophores generally absorb light at a wavelength which is usually at least 10 nm higher (such as at least 20 nm higher) than the maximum absorbance wavelength of the donor fluorophore, and have a fluorescence emission maximum at a wavelength ranging from about 400 to 900 nm. Acceptor fluorophores have an excitation spectrum which overlaps with the emission of the donor fluorophore, such that energy emitted by the donor can excite the acceptor. An acceptor fluorophore may be attached to a nucleic acid molecule.
In embodiments, an acceptor fluorophore is a quencher, such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACK HOLE QUENCHERS™ (Glen Research), ECLIPSE™ Dark Quencher (Epoch Biosciences), or IOWA BLACK™ (Integrated DNA Technologies). A quencher can reduce or quench the emission of a donor fluorophore. In such an example, instead of detecting an increase in emission signal from the acceptor fluorophore when in sufficient proximity to the donor fluorophore (or detecting a decrease in emission signal from the acceptor fluorophore when a significant distance from the donor fluorophore), an increase in the emission signal from the donor fluorophore can be detected when the quencher is a significant distance from the donor fluorophore (or a decrease in emission signal from the donor fluorophore when in sufficient proximity to the quencher acceptor fluorophore).
Donor Fluorophores (sometimes simply referred to as a “fluorophore”) are fluorophores or luminescent molecules capable of transferring energy to an acceptor fluorophore, thereby generating a detectable fluorescent signal from the acceptor. Donor fluorophores are generally compounds that absorb in the range of about 300 to 900 nm, for example about 350 to 800 nm. Donor fluorophores have a strong molar absorbance coefficient at the desired excitation wavelength, for example greater than about 103 M−1 cm−1.
A donor fluorophore may be attached to a nucleic acid molecule. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987).
In embodiments, a probe includes at least one fluorophore, such as an acceptor fluorophore or donor fluorophore. For example, a fluorophore can be attached at the 5′- or 3′-end of the probe. In specific examples, the fluorophore is attached to the base at the 5′-end of the probe, the base at its 3′-end, the phosphate group at its 5′-end or a modified base, such as a T internal to the probe.
Probes are generally at least 20 nucleotides in length, such as at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, or more contiguous nucleotides complementary to the target nucleic acid molecule, such as 20-60 nucleotides, 20-50 nucleotides, 20-40 nucleotides, or 20-30 nucleotides.
An exemplary pair of primers for a conserved bacterial 16S target nucleic acid sequence have the following sequences:
In embodiments, a primer has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these primer sequences.
Exemplary probes for an amplicon amplified from a conserved bacterial 16S target nucleic acid sequence have the following sequences/structures:
In embodiments, a probe has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these probe sequences.
An exemplary pair of primers for an E. coli target nucleic acid sequence have the following sequence:
In embodiments, a primer has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these primer sequences.
An exemplary probe for an amplicon cmplified from the E. coli target nucleic acid sequence has the following sequence/structure:
In embodiments, a probe has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to this probe sequence.
An exemplary pair of primers for a Salmonella target nucleic acid sequence have the following sequence:
In embodiments, a primer has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these primer sequences.
An exemplary probe for an amplicon cmplified from the Salmonella target nucleic acid sequence has the following sequence/structure:
In embodiments, a probe has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to this probe sequence.
Exemplary pairs of primers for PsiK, PsiM, PsiH, and PsiD target nucleic acid sequences have the following sequences:
In embodiments, a primer has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these primer sequences.
Exemplary probes for amplicons amplified from PsiK, PsiM, PsiH, and PsiD target nucleic acid sequences have the following sequences/structures:
In embodiments, a probe has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these probe sequences.
An exemplary pair of primers for a fungal ITS region have the following sequences:
In embodiments, a primer has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to one of these primer sequences.
An exemplary probe for an amplicon amplified from fungal ITS region has the following sequence/structure:
In embodiments, a probe has a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to this probe sequence.
To design primers and probes, it may be useful to perform sequence alignments of nucleic acid (e.g., gene) regions to identify target nucleic acid sequences based on which regions of the nucleic acid (e.g., gene) of interest are polymorphic (variable) or conserved. Such sequence alignments may be performed using multiple different Psilocybe strains/species/samples (e.g., at least 10, 20, 30, 40, 50, 60, 70, or 80 different Psilocybe strains/species/samples).
In embodiments, a primer or probe is designed so as to hybridize to a region that is conserved such that the region has less than 3%, 2%, 1%, or 0.5% genomic variation in a sequence alignment. In embodiments, any or all target nucleic acid sequences are regions that have less than 3%, 2%, 1%, or 0.5% genomic variation in a sequence alignment.
Due to polymorphism, primers and a probe were not designed based on the sequence alignment of
Similar sequence alignments may be helpful for designing primers and probes that hybridize to any of the target nucleic acid sequences of the methods discussed above and the kits discussed below.
Kits for performing any of the processes described herein constitute other embodiments. In general, the kits include the primers and/or probes as described herein. The kit may also contain other suitably packaged reagents and other materials needed for the particular assay protocol, for example, controls and polymerizing agents, as well as instructions for conducting the assay/test.
In use, the components of the PCR kit, when applied to the sample, create a reagent mixture which enables the amplification and detection of the target nucleic acid sequences described herein. The reagent mixture thus includes the components of the kit as well as the sample (once provided) which may contain the target nucleic acids of interest.
In embodiments, a kit for detecting contamination in a Psilocybe sample comprises (i) primers for amplifying a bacterial target nucleic acid sequence, a Psilocybe target nucleic acid sequence, and/or a fungal target nucleic acid sequence, and (ii) probes for detecting the amplicons amplified from any of those target nucleic acid sequences. For instance, the kit may comprise primers for amplifying a bacterial target nucleic acid sequence and a Psilocybe target nucleic acid sequence and probes for detecting amplicons of the bacterial target nucleic acid sequence and the Psilocybe target nucleic acid sequence. The probes may include a fluorescent label. The kit may further comprise reagents including any of a lysis buffer, magnetic beads, a binding buffer, a wash solution, an elution solution, a DNA polymerase, or dNTPs. The kit may also further comprise instructions for performing an assay (or a test) to detect contamination in a Psilocybe sample.
In embodiments, the bacterial target nucleic acid sequence is a 16S target nucleic acid sequence and the Psilocybe target nucleic acid sequence is any of a PsiK, PsiM, PsiH, or PsiD target nucleic acid sequence. That is, the Psilocybe target nucleic acid sequence may be any combination of PsiK, PsiM, PsiH, or PsiD target nucleic acid sequences. For example, the Psilocybe target nucleic acid sequences may include a target nucleic acid sequence from each of PsiK and PsiM or may include a target nucleic acid sequence from each of PsiK, PsiM, and PsiD.
In embodiments, the kit comprises primers for amplifying a fungal target nucleic acid sequence and a probe for detecting amplicons of the fungal target nucleic acid sequence. The fungal target nucleic acid sequence may be an Internal Transcribed Spacer (ITS) target nucleic acid sequence.
In embodiments, the probes have different fluorescent labels based on different amplicons sequences to which the probes hybridize. The probes may have a probe structure that includes a fluorophore and a quencher.
In embodiments, the primers include a primer having a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOS: 8-21. In embodiments, the probes include a probe having a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOS: 22-29.
In embodiments, the bacterial and Psilocybe target nucleic acid sequences include a target nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 1-6. In embodiments, the fungal target nucleic acid sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, disclosed herein are incorporated by reference in their entirety, particularly for the disclosure referenced herein.
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting in any way. That is, the following examples are provided to illustrate various inventive aspects. They are not intended to be limiting.
DNA isolation from P. cubensis spores: Spores were obtained from 4 vendors (Sporeworks.com, Premiumspores.com, Mushroom.com and InnoculateTheWorld.com).
Briefly, 1.4 ml of spores were centrifuged, decanted and resuspended in 200 ul of ddH20. 25 μl of a Thaumatin-like protein was added and incubated at 37° C. (Medicinal Genomics part #420206) for 30 minutes. 12.5 μl of MGC lysis buffer was added and incubated at 65° C. for 30 minutes with 9 steel beads. Vortexing was performed every 7 minutes. Lysed sample were micro-centrifuged and 200 μl of supernatant was aspirated and added to 250 μl of Medicinal Genomics (MGC) binding buffer (MGC part #420001) for magnetic bead isolation. The samples were incubated with the MGC magnetic bead mixture for 10 minutes, magnetically separated and washed two times with 70% ethanol. The beads were dried at 37° C. for 5 minutes to remove excess ethanol and eluted with 25 μl of ddH20.
Real time PCR (qPCR): qPCR was performed using 5 μL of DNA (3 ng/μL) 12.5 μL 2× LongAmp (NEB) with 1.25 μL of 10 μM 16S primers/probe and/or ITS primers/probe (i.e., MGC-ITS3F and MGC-ITS3R primers) as well as primers/probes for PsiK, PsiM, and/or PsiD. 10 μL ddH20 was used for a 25 μL total reaction volume. An initial 95° C. 5-minute denaturation step was performed followed by 40 cycles of 95° C. for 15s and 65° C. for 90s.
The following primers and probes were used:
The qPCR results are shown in
This application claims the benefit of U.S. Provisional Application No. 63/285,609, filed on Dec. 3, 2021. The disclosure of the prior application is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/080719 | 12/1/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63285609 | Dec 2021 | US |
