Patent Application
20220396828
The present invention relates to primer pair and an oligonucleotide probe for hybridizing to at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, an oligonucleotide set for use in a nucleic acid amplification process or a nucleic acid detection process for determining the presence of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group in a sample, a kit for detecting the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in a sample, and a method of determining the presence or absence of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group in a sample.
Clostridioides difficile (synonym “C. difficile”, previously named Clostridium difficile, Non-Patent literature 1) is a sporulating Gram-positive bacillus producing a pathogenic toxin mediating intestinal diseases in humans and animals. Dependent on the age of the patient, intestinal flora and the Clostridioides difficile strain the clinical outcome of a Clostridioides difficile infection (CDI) may be asymptomatic or may range to severe disease syndromes, such as diarrhea, abdominal pain, fever and leukocytosis.
Meanwhile, Clostridioides difficile is recognized as a main cause for infectious diarrhea in hospitalized patients or patients undergoing a treatment damaging the normal intestinal flora, such as antibiotic or anticancer treatment. Clostridioides difficile may, as a consequence of the damage to the intestinal flora produced by the mentioned treatments overgrow and produce toxin A, toxin B and binary toxin CDT, which develop symptoms, such as diarrhea. Clostridioides difficile is excreted in feces from infected individuals, and transfection to other individuals may occur orally, in particular by the mucosa of the mouth by colonized hands or instruments.
Currently, a Clostridioides difficile strain known as type B1 by Restriction Endonuclease Analysis, North American (NA) pulsed-field type 1 (NAP1) by Pulsed-field Gel Electrophoresis, and 027 by PCR-ribotyping (B1/NAP1/027) is regarded the single most important epidemic strain causing Clostridioides difficile-associated disease in North America and Europe (Non-Patent Literature 2).
Thus, there is a need to determine the presence of hyper-virulent Clostridioides difficile, in particular Clostridioides difficile B1/NAP1/027 strains.
Presently, the detection methods mainly focus on identification of toxin A, toxin B and binary toxin CDT in samples, preferably stool samples.
Testing methods include tissue culture cytotoxicity assays and/or Clostridioides difficile culture identification and/or enzyme immunoassays (EIAs) for toxins A and/or B, and EIAs for glutamate dehydrogenase (GDH, antigen of the C. difficile cell wall). Whereas the tissue culture cytotoxicity assay and culture identification are labor and time consuming, as the results are only obtained within 24 to 96 hours, EIA tests results show comparatively low specificity.
Direct stool culture and cell cytotoxin neutralization assay (CCNA) tests are currently the reference standard methods for the diagnosis of CDI. However, both methods are laborious, time consuming, and must be performed by qualified, trained personnel.
Recently, polymerase chain reaction tests have been made available. These methods detect relevant genes with high sensitivity, specificity, and low turnaround time. One test relates to the use of a set of primers that can detect a set of three genes, namely tcdA and tcdB, which respectively code toxins A and B, as well as tcdC, which is present in mutated form in hyper-virulent Clostridioides difficile strains, and so allows overexpression of toxin A and B (Patent Document 1). This method enables the detection of hyper-virulent Clostridioides difficile strains, in particular Clostridioides difficile strains belonging to the B1/NAP1/027 group. However, the test requires many process steps and the use of different laboratory equipment, which makes its set up complex, expensive and time consuming.
Commercially available products for Clostridium difficile diagnosis include the BD GeneOhm Cdiff (BD Diagnostics; San Diego, Calif., USA), Prodesse ProGastro Cd (Gen-Probe Inc.; San Diego, Calif., USA), Xpert Clostridium difficile (Cepheid; Sunnyvale, Calif., USA), and illumigene Clostridium difficile (Meridian Biosciences; Cincinnati, Ohio, USA) real-time PCR tests. All the tests target the tcdB gene, except for the illumigene Clostridium difficile test, which targets the tcdA gene. The Xpert Clostridium difficile/Epi (Cepheid) is additionally capable of detecting the binary toxin. Compared to the CCNA and/or toxigenic culture, the PCR assays were reported with enhanced sensitivities and specificities ranging from 77% to 100% and 93% to 100%, respectively (Non-Patent Literature 3).
Nevertheless, there still exists a need to provide an alternative method to determine the presence of a hyper-virulent Clostridioides difficile strain, in particular a hyper-virulent Clostridioides difficile strains belonging to the B1/NAP1/027 group, wherein the method preferably exhibits a suitable sensitivity and/or specificity, is simple, time and/or cost saving.
Thus, the problem addressed by the present invention is to provide a method for determining the presence of hyper-virulent Clostridioides difficile strains belonging to the B1/NAP1/027 group, preferably with a suitable specificity and/or sensitivity and more preferably being simple, time and/or cost saving.
One or more problems addressed by the present invention is/are solved by the subject matter of the independent claims, namely the primer pair or oligonucleotide probe for hybridizing to at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain belonging to the B1/NAP1/027 group, the oligonucleotide set comprising the primer pair or oligonucleotide probe, the kit for detecting the presence of a hyper-virulent strain of Clostridioides difficile belonging to the B1/NAP1/027 group in a sample comprising the primer pair or oligonucleotide probe, and the method for determining the presence of a hyper-virulent Clostridioides difficile strain belonging to the B1/NAP1/027 group. Advantages (preferred embodiments) are set out in the detailed description hereinafter and/or the accompanying figures as well as in the dependent claims.
Accordingly, a first aspect of the present invention relates to a primer pair for hybridizing to at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, characterized in that the primer pair comprises or consists of
The primer pair according to the first inventive aspect is generally used for hybridizing to and amplifying of at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, preferably of a chloramphenicol acetyltransferase gene according to SEQ ID No. 3, more preferably of a chloramphenicol acetyltransferase gene according to SEQ ID No. 4.
A second aspect of the present invention relates to an oligonucleotide probe for hybridizing to at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, characterized in that the oligonucleotide probe comprises or consists of an isolated oligonucleotide a) comprising or consisting of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein the oligonucleotide comprises at least 15 nucleotides, or an isolated oligonucleotide with at least 15 nucleotides comprising or consisting of a complementary oligonucleotide sequence to the isolated oligonucleotide comprising or consisting of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1 and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 15 and 25 nucleotide residues, preferably 20 to 25 nucleotide residues.
The oligonucleotide probe according to the second inventive aspect is generally used for hybridizing to and detecting of at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, preferably of a chloramphenicol acetyltransferase gene according to SEQ ID No. 3, more preferably of a chloramphenicol acetyltransferase gene according to SEQ ID No. 4. The oligonucleotide probe may be any suitable probe, such as a hybridization probe, hydrolysis probe, etc.
A third aspect of the present invention relates to an oligonucleotide set for use in a nucleic acid amplification process or a nucleic acid detection process for determining the presence of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group in a sample, characterized in that the oligonucleotide set comprises the inventive primer pair of the first inventive aspect or the inventive oligonucleotide probe of the second inventive aspect, and comprises one or more additives.
A fourth aspect of the present invention relates to a kit for detecting the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in a sample comprising the inventive primer pair of the first inventive aspect, the inventive oligonucleotide probe of the second inventive aspect and/or the inventive oligonucleotide set of the third inventive aspect, characterized in that the oligonucleotide a) and oligonucleotide b) of the primer pair or the oligonucleotide a) of the oligonucleotide probe hybridize under stringent conditions to at least part of SEQ ID No. 3.
A fifth aspect of the present invention relates to a method of determining the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in a sample, the method comprises or consists of:
The inventive aspects of the present invention as disclosed hereinbefore can comprise any possible (sub-)combination of the preferred inventive embodiments as set out in the dependent claims or as disclosed in the following detailed description and/or in the accompanying figures, provided the resulting combination of features is reasonable to a person skilled in the art.
Further characteristics and advantages of the present invention will ensue from the accompanying drawings, wherein
The inventors of the present invention have found out that the inventive primer pair of the first inventive aspect and the inventive oligonucleotide probe of the second inventive aspect specifically enable to determine the presence of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, whereas strains closely related to B1/NAP1/027 as well as other strains are truly not determined. The specificity is due to the fact that the inventive primer pair and the inventive oligonucleotide probe have been optimized to specifically amplify and detect respectively such a part of the chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 as target, which is specific for hyper-virulent Clostridioides difficile strains of the B1/NAP1/027 group and is not present in other closely related strains. Thus, the inventive primer pair and the inventive oligonucleotide probe show a high specificity for Clostridioides difficile strains belonging to the B1/NAP1/027 group. Furthermore, the inventive detection method of the fourth inventive aspect provides a simple and highly sensitive detection method for hyper-virulent Clostridioides difficile strains, more preferably hyper-virulent Clostridioides difficile strains belonging to the B1/NAP1/027 group (for detailed information see Example section).
According to a preferred embodiment of all aspects of the present invention, the oligonucleotide coding the chloramphenicol acetyltransferase gene in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain comprises or consists of SEQ ID No. 3. The oligonucleotides represented in SEQ ID No. 3 refer to the set of oligonucleotides representing the full chloramphenicol acetyltransferase gene in the transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain. According to a further preferred embodiment of the first inventive aspect, the oligonucleotides coding the chloramphenicol acetyltransferase gene in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain comprise or consist of SEQ ID No. 4, as this sequence is specific for Clostridioides difficile strains belonging to the B1/NAP1/027 group.
In the context of the present invention, the expression “an additionally or alternatively further preferred embodiment” or “an additionally or alternatively preferred embodiment” or “an additional or alternative way of configuring this preferred embodiment” means that the feature or feature combination disclosed in this preferred embodiment can be combined in addition to or alternatively to the features of the inventive subject matter including any preferred embodiment of each of the inventive aspects, provided the resulting feature combination is reasonable to a person skilled in the art.
As already mentioned in the summary section of the invention, the inventive primer pair according to the first inventive aspect is generally used for hybridizing to and amplifying of at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, whereas the inventive oligonucleotide probe according to the second inventive aspect is generally used for hybridizing to and detecting of at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group.
The isolated oligonucleotide a) of the inventive primer pair of the first inventive aspect or the inventive oligonucleotide probe of the second inventive aspect comprises or consists of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein the oligonucleotide comprises at least 10 nucleotides and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 25 nucleotide residues. Alternatively, an isolated oligonucleotide with at least 10 nucleotides can be used as oligonucleotide a), which comprises or consists of a complementary oligonucleotide sequence to the oligonucleotide comprising or consisting of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 25 nucleotide residues. In order to increase the hybridization accuracy, the isolated oligonucleotide a) may comprise 15 or more, more preferably 20 or more, preferably 22 or more, alternatively 25 to 50 nucleotides.
In the context of the present invention, the term “nucleotide”/“nucleotides” and “nucleotide residue”/“nucleotide residues” are used synonymously.
In the context of the present invention, the term “alignment” refers to a result of matching up nucleotide residues of two sequences so as to achieve a maximal level of identity, wherein the alignment is an ungapped sequence alignment. In particular, for calculating the sequence identity the alignment of oligonucleotide a) and SEQ ID No. 1 is used, which means that the respective nucleotide residue sequence of oligonucleotide a) is matched up with the corresponding nucleotide residue sequence of SEQ ID No. 1. For calculating the sequence identity using the alignment of oligonucleotide b) and SEQ ID No. 2, the respective nucleotide residue sequence of oligonucleotide b) is matched up with the corresponding nucleotide residue sequence of SEQ ID No. 2.
In the context of the present invention, the expression “total number of nucleotide residues in the alignment” (synonymous: “alignment length”) means the number of aligned matching nucleotide residues and intervening non-matching nucleotide residues of one of the two aligned sequences. The respectively aligned nucleotide residues form respectively corresponding nucleotide residue pairs. The “total number of nucleotide residues in the alignment”/the “alignment length” is used as denominator in sequence identity calculation. The nucleotide residues in the respective corresponding nucleotide residue pairs can respectively show identity of the nucleotide residues (synonym: “matching nucleotide residues”) or non-identity of the nucleotide residues.
In the context of the present invention, the expression “matching nucleotide residues” or “identity of nucleotide residues” means that the nucleotide residues of the respective nucleotide residue pair represent either identical nucleotide residues or identical nucleotide residue analogs, such as Locked Nucleic Acid (LNA) and DNA.
The isolated oligonucleotide a) of the inventive oligonucleotide probe of the second inventive aspect comprises or consists of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein the oligonucleotide comprises at least 15 nucleotides and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 25 nucleotide residues. Alternatively, an isolated oligonucleotide having at least 15 nucleotides can be used as oligonucleotide a), which comprises or consists of a complementary oligonucleotide sequence to the oligonucleotide comprising or consisting of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 25 nucleotide residues. In order to increase the hybridization accuracy, the isolated oligonucleotide a) may comprise 20 or more, preferably 22 or more, alternatively 25 to 50 nucleotides.
In the context of the present invention the expressions “oligonucleotide a) comprises or consists of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein the oligonucleotide comprises at least 10 nucleotides” and “oligonucleotide a) comprises or consists of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein the oligonucleotide comprises at least 15 nucleotides” respectively mean that the oligonucleotide a) comprises a sequence with at least 10/15 nucleotides and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10/15 and 25 nucleotide residues. This understanding follows from the use of the wording “comprises or consists of at least 88%” according to which follows, that the oligonucleotide a) comprises a sequence having at least 88% sequence identity or consists of a sequence having at least 88% sequence identity. Furthermore, the wording “sequence identity to SEQ ID No. 1” requires in view of the claimed length of the oligonucleotide a) of at least 10/15 nucleotides, which may thus be less or more than the length of 25 nucleotides of SEQ ID No. 1, that the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10/15 and 25 nucleotide residues. The same understanding relates to the isolated oligonucleotide to be used as oligonucleotide a) comprising at least 10/15 nucleotides, which comprises or consists of a complementary oligonucleotide sequence to the oligonucleotide comprising or consisting of at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide a) and the SEQ ID No. 1 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10/15 and 25 nucleotide residues.
The second isolated oligonucleotide b) of the inventive primer pair is generally selected from primer oligonucleotides hybridizing to at least a portion of the chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group, preferably of SEQ ID No. 3, more preferably SEQ ID No. 4. More preferably, the second isolated oligonucleotide b) of the inventive primer pair comprises or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2, wherein the oligonucleotide comprises at least 10 nucleotides and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide b) and the SEQ ID No. 2 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 21 nucleotide residues. In order to increase the hybridization accuracy, the isolated oligonucleotide b) may preferably comprise 15 or more, more preferably 20 to 50 nucleotides.
In the context of the present invention the expression “oligonucleotide b) comprises or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2, wherein the oligonucleotide comprises at least 10 nucleotides” means that the oligonucleotide b) comprises a sequence with at least 10 nucleotides and wherein the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide b) and the SEQ ID No. 2 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 21 nucleotide residues. This understanding follows from the use of the wording “comprises or consists of at least 90%” according to which follows, that the oligonucleotide b) comprises a sequence having at least 90° % sequence identity or consists of a sequence having at least 90° % sequence identity. Furthermore, the wording “sequence identity to SEQ ID No. 2” requires in view of the claimed length of the oligonucleotide b) of at least 10 nucleotides, which may thus be less or more than the length of 21 nucleotides of SEQ ID No. 2, that the sequence identity is respectively calculated as ratio of a number of matching nucleotide residues of an alignment between the oligonucleotide b) and the SEQ ID No. 2 to a total number of nucleotide residues in the alignment, wherein the total number of nucleotide residues in the alignment is between 10 and 21 nucleotide residues.
According to an exemplified embodiment, the inventive primer pair comprises or consists of an isolated oligonucleotide a) according to SEQ ID No. 1 and an isolated oligonucleotide b) according to SEQ ID 2.
According to an additionally or alternatively further preferred embodiment of the first inventive aspect, the oligonucleotide a) and/or oligonucleotide b) of the inventive primer pair may independently from each other comprise one or more added functionalities, e.g. quencher and/or fluorophores in particular detectable by fluorescence resonance energy transfer (FRET), radioisotopes and/or may comprise one or more modified nucleotides, e.g. locked nucleic acid (LNA). Such added functionalities may enhance the hybridization of the inventive primer pair to the target and/or detection of the inventive oligonucleotide probe.
According to an exemplified embodiment, the inventive oligonucleotide probe comprises or consists of an isolated oligonucleotide a) according to SEQ ID No. 1 or its complementary sequence and one or more suitable detection functionalities for detection of Clostridioides difficile of the B1/NAP1/027 group, in particular for quantitative detection of Clostridioides difficile of the B1/NAP1/027 group, such as quencher and/or fluorophores moieties, more preferably a fluorophore moiety and a quencher moiety at the 5′ end and at the 3′ end of the oligonucleotide probe respectively.
The genome of a reference strain R20291 of Clostridioides difficile of the B1/NAP1/027 group has been chosen as an example in order to optimize the inventive primer pair of the first inventive aspect and the inventive oligonucleotide probe of the second inventive aspect. The fully sequenced genome is deposited with gene accession no: FN545816.1.
As set out in detail in the example section (executive examples 1 and 2), the inventive primer pair facilitates to specifically hybridize to and to amplify hyper-virulent Clostridioides difficile strains of the B1/NAP1/027 group (group 1), whereas the inventive primer pair does not hybridize to and amplify strains closely related to B1/NAP1/027 as determined by ribotyping (group 2) as well as other Clostridioides difficile strains, such as 630 (group 3). Thus, the inventive primer pair is highly specific for the determination of hyper-virulent Clostridioides difficile strains of the B1/NAP1/027 group in comparison to closely related strains of group 2 or other strains of group 3.
The same is true for the inventive oligonucleotide probe, which facilitates to specifically hybridize to and to detect hyper-virulent Clostridioides difficile strains of the B1/NAP1/027 group (group 1), whereas the inventive oligonucleotide probe does not hybridize to and detect strains closely related to B1/NAP1/027 as determined by ribotyping (group 2) as well as other Clostridioides difficile strains, such as 630 (group 3). Thus, the inventive oligonucleotide probe is highly specific for the detection of hyper-virulent Clostridioides difficile strains of the B1/NAP1/027 group in comparison to closely related strains of group 2 or other strains of group 3.
All features and embodiments disclosed with respect to the first and second aspects of the present invention are combinable alone or in (sub-)combination with each other and with the third to fifth aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a skilled person.
According to the third aspect of the present invention, an oligonucleotide set is provided for use in a nucleic acid amplification process for determining the presence of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group in a sample, characterized in that the oligonucleotide set comprises the inventive primer pair of the first inventive aspect or the inventive oligonucleotide probe of the second inventive aspect, and one or more solvents. Suitable solvents are readily available in the prior art.
All features and embodiments disclosed with respect to the third aspect of the present invention are combinable alone or in (sub-)combination with each of the first, second, fourth and fifth aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a skilled person.
According to the fourth aspect of the present invention a kit for detecting the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in a sample comprising the inventive primer pair according to the first inventive aspect, the inventive oligonucleotide probe according to the second inventive aspect and/or the oligonucleotide set according to the third inventive aspect, characterized in that the inventive oligonucleotide a) and oligonucleotide b) of the primer pair or the oligonucleotide a) of the oligonucleotide probe respectively hybridize under stringent conditions to at least part of SEQ ID No. 3, preferably at least part of SEQ ID No. 4. For example, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is too stringent, no product will be produced. The stringency of an amplification reaction is, thus, routinely modulated and adjusted by the skilled person in order to affect the outcome of the amplicon profile, in particular by altering variables, such as reagent concentrations, cycling conditions.
According to an additionally or alternatively preferred embodiment the inventive kit for detecting the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in a sample further comprises one, two, three, four or more constituents (reactants) selected from the group of enzymes, such as polymerase (e.g. taq DNA polymerase), ligase, helicase, or recombinase; deoxynucleotides; control template; buffer and reaction additives. Preferably, the buffer is dissolved in water, preferably nuclease free water, and comprises or consists of one or more constituents selected from the group of salts, such as potassium chloride (KCl), ammonium sulfate (NH4)2SO4, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), or proteins, such as albumin including Bovine Serum Albumin; and/or the reaction additives comprise or consist of one, two or more constituents selected from the group of co-factors, such as magnesium chloride (MgCl2); dimethyl sulfoxide (DMSO); fluorophores, such as fluorescent dyes intercalating into double stranded nucleic acid strands (e.g. SYBR Green I); and quenchers.
One or more of the constituents in the inventive kit may be present in a concentrated form, i.e. need to be diluted, preferably with nuclease free water, prior to their application in the amplification reaction. As an example one or more of the constituents may be present in a two-fold concentrated form, i.e. the respectively concentrated constituents need to be diluted to twice of the concentrated volume.
As an example, the stringent condition in the inventive kit for detecting the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group comprises nuclease free water and 0.05 to 0.1 U/μl polymerase, preferably taq DNA polymerase, 10 to 200 mM Tris-HCl, 0.5 to 5 mM, preferably 3 mM MgCl2, 50 to 500, preferably 50 mM KCl and a pH in the range of 6.8 to 9.1, preferably pH of 8.4 to 8.8. In addition, a suitable amount of detection agent, such as fluorophore, preferably a fluorescent dye intercalating into double stranded DNA is also present in the kit.
All features and embodiments disclosed with respect to the fourth aspect of the present invention are combinable alone or in (sub-)combination with each of the first, second, third and fifth aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a skilled person.
According to the fifth aspect of the present invention, a method of determining the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in a sample is provided. As already mentioned above, the inventive detection method provides a simple, and thus, cost effective detection method that exhibits at the same time high sensitivity.
The inventive method comprises step a) performing a nucleic acid hybridization and amplification reaction comprising a denatured nucleic acid extract of the sample as a template and an inventive primer pair or the inventive oligonucleotide set or the inventive kit, wherein the oligonucleotide set and the kit respectively comprise the primer pair, in order to generate an amplicon of at least a portion of a chloramphenicol acetyltransferase gene coded in a transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group. The denatured nucleic acid extract of the sample may be commercially available or may be produced from the specimen sample according to suitable methods of the prior art, such as described as “Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing” (James Heng Chiak Sim et al., Journal of Clinical Microbiology, July 2015, Vol. 53, Number 7, page. 2329-2331). Any suitable kit may be used for DNA isolation, such as Genomic Mini Kit (A&A Biotechnology). Diagnosis and typing of human Clostridioides difficile infection may be conducted by suitable standard operating procedures (SOPs), such as “Laboratory procedures for diagnosis and typing of human Clostridium difficile infection” issued by the European Center for Disease Prevention and Control 19 Oct. 2018.
According to an additional or alternative preferred embodiment, the amplification reaction is selected from the group consisting of a polymerase chain reaction (PCR), such as Real-Time PCR (qPCR), a ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex PCR-ligase detection reaction assay, multiplex ligation dependent probe amplification (MLPA), multiplex oligonucleotide ligation PCR (MOL-PCR), Isothermal DNA amplification, Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HAD), Signal-Mediated Amplification of RNA Technology (SMART), Recombinase Polymerase Amplification (RPA), Polymerase Spiral Reaction (PSR), Strand displacement amplification. In particular, real-Time PCR (qPCR) not only allows qualitative, but also quantitative detection of the hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group in the sample.
The ligase chain reaction (LCR) is in particular suitable in case in addition to the inventive primer pair, a second primer pair for a second template strand is used.
The ligase detection reaction (LDR) is a modified LCR technique, where only two oligonucleotides, instead of four oligonucleotides bind adjacently on one target strand.
With respect to multiplex ligation dependent probe amplification (MLPA) two oligonucleotide probes bind adjacently on a target sequence and are ligated only in presence of their complementary target DNA.
Multiplex oligonucleotide ligation PCR (MOL-PCR) is similar to MLPA, but a flow cytometric device is used to identify each bead to measure fluorescence intensity for each bead-probe complex. For MOL-PCR the reverse primer and not the forward primer is fluorescently labelled.
Rolling Circle Amplification (RCA) is an anisothermal enzymatic process that uses DNA or RNA polymerases to produce single stranded DNA (ssDNA) or RNA molecules, which are a connection in series of complementary units of a template.
Helicase-Dependent Amplification (HAD) follows replication fork mechanism for amplification, which utilizes DNA helicase for unwinding activity Helicase separates the duplex DNA into single-stranded DNA for in vitro amplification of target DNA. The amplification is extended by exonuclease deficient DNA polymerases at a single constant temperature. The HAD products are detected by gel electrophoresis.
Signal-Mediated Amplification of RNA Technology (SMART) is based on signal amplification and it does not depend on copying of target sequences. The SMART method consists of two single-stranded oligonucleotide probes that are annealed to a specific target sequence and then it forms a three-way junction (3WJ) structure. The reaction is carried out by three enzymes like Bst DNA Polymerase, T7 RNA polymerase and DNA polymerase at a constant temperature of 41° C. The amplicons are detected by ELOSA and real-time.
Recombinase Polymerase Amplification (RPA) is an isothermal method and amplifies target DNA sequences by using a DNA polymerase, recombinase and DNA-binding proteins at a temperature of 37-42° C. The amplified products are detected by the fluorophore, quencher groups and microfluidic devices.
Polymerase Spiral Reaction (PSR) can be finished within 1 h with a high sensitivity (up to 109 copies) and high specificity to detect the target. The PSR method exploits the advantages of PCR in which only one pair of primers is needed and isothermal amplification techniques like LAMP assay.
Strand displacement amplification is based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo-klenow) to extend the 3′-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa.
The inventive determination method further comprises as step b) detecting the amplicon generated in step a). According to an additional or alternative preferred embodiment, the amplicon is detected by use of a non-specific detection method, such as detection of fluorescence with suitable means. As an example, a fluorescence dye non-specifically intercalating into the double stranded nucleic acid strand of the amplicon may be detected. SYBR Green I, is an example of a commonly used fluorescent DNA binding dye, which binds all double-stranded DNA and detection is monitored by measuring the increase in fluorescence throughout the cycle. SYBR Green I has an excitation and emission maxima of 494 nm and 521 nm, respectively. Specificity of Sigma's SYBR based QPCR detection is greatly enhanced by the incorporation of a hot-start mediated taq polymerase, JumpStart Taq. Alternatively, fluorophores and quenchers as FRET pairs, e.g. in TaqMan assays or in techniques using hybridization probes can be used. As an example, the inventive oligonucleotide probe of the second inventive aspect can be used to detect the generated amplicon of step a).
The method further comprises as step c) determining the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in the sample, in case the amplicon is positively detected in step b) determining the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group in the sample as a function of the detected amplicon in step b). In other words, in case the amplicon of at least a portion of a chloramphenicol acetyltransferase gene coded in the transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group is generated in step a) and detected in step b), then the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group is determined in step c). This means in turn, that in case the amplicon of at least a portion of a chloramphenicol acetyltransferase gene coded in the transposon of family CTn-027 of a hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group is not generated in step a) and, thus, not detected in step b), then the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group is not determined in step c). As set out in the example section, the inventive method for determining the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group is highly specific. This means, that the inventive primer pair, when subjected to Clostridioides difficile strains closely related to the B1/NAP1/027 group or other strains, do not generate the amplicon in step a) and, thus, those strains are truly not determined as Clostridioides difficile of the B1/NAP1/027 group. Moreover, in view of the use of the amplification reaction, the inventive method is also highly sensitive as well as simple, time and cost effective.
In general, the sample to be detected by the inventive method of the fifth inventive aspect may be any suitable specimen. According to an additional or alternative preferred embodiment, the sample for determining the presence of a hyper-virulent strain of Clostridioides difficile of the B1/NAP1/027 group is selected from the group consisting of a human sample, an animal sample, an object sample or a food sample, more preferably selected from stool sample, a smear test sample, a body fluid sample, or a tissue sample, respectively from human or animal.
All features and embodiments disclosed with respect to the fifth aspect of the present invention are combinable alone or in (sub-)combination with each of the first, second, third and fourth aspects of the present invention respectively including each of the preferred embodiments thereof, provided the resulting combination of features is reasonable to a skilled person.
Further characteristics and advantages of the present invention will ensue from the following description of example embodiments of the inventive aspects with reference to the accompanying drawings.
All of the features disclosed hereinafter with respect to the example embodiments can alone or in any sub-combination be respectively combined with features of the different aspects of the present invention including features of preferred embodiments thereof, provided the resulting feature combination is reasonable to a person skilled in the art.
To show specificity of the inventive primer pair and inventive oligonucleotide probe, the following alignment was conducted. First, the target sequence of the chloramphenicol acetyltransferase gene according to SEQ ID No. 3 as present in Clostridioides difficile strain R2091 complete genome was entered into Basic Local Alignment Search Tool (BLAST) in order to align target sequence of Clostridioides difficile of the B1/NAP1/027 group to any known homologues. The results obtained from the BLAST alignment were grouped as follows:
Group 1 as set out in table 1 below contains strains that have 100% query cover and identity with target sequence, all strains are recognized as specimens that belong to Clostridioides difficile of the B1/NAP1/027 group and are deemed to exhibit true positive scores in detection step b) of the inventive method.
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain DSM
Clostridioides
difficile strain DSM
Clostridioides
difficile strain DSM
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridioides
difficile strain
Clostridium difficile
Clostridium difficile
Clostridium difficile
Clostridium difficile
Group 2 as set out in table 2 below contains strains with lower coefficient in query cover and identity, hence has some difference in sequence, such as deletions or inversions, all strains are recognized as specimens that do not belong to Clostridioides difficile of the B1/NAP1/027 group, but are deemed to be closely related. This group has been chosen, as it was expected that they might produce false positive scores in the detection step b) of the inventive method.
Clostridioides
difficile strain
Clostridioides
difficile strain DSM
Clostridioides
difficile strain
Clostridioides
difficile strain AK
Clostridioides
difficile strain Z31
Clostridioides
difficile ATCC
Clostridium difficile
Clostridioides
difficile strain DSM
Group 3 comprises all other strains, such as Clostridium difficile 630 strain, that do not show homology to the target sequence.
Protocol of Sample Preparation:
1. C. difficile ATCC 9689 bacteria were grown on Columbia Agar with 5% Sheep Blood plates (Oxoid; catalog no. PB5039A) at 37° C. under anaerobic conditions (AnaeroGen Compact, Oxoid; catalog no. AN0010C and AN0020D) for 48 hours.
2. C. difficile colonies were scraped from plates with inoculating loop and suspended in 10 mM Tris-HCl pH 8.0 buffer to equal 1.0 McFarland.
3. For DNA extraction a 100 μl volume of a 1.0 McFarland suspension per sample were used.
4. Bacterial DNA was extracted using a Genomic Mini Kit (A&A Biotechnology) according to the manufacturer protocol.
In analogy, the sample preparation is conducted with respect to C. difficile 630 strain. With respect to C. difficile R20291 strain a DNA source was provided as sample material.
Respective Bacteria culture and/or DNA material can be obtained from Public Health England, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, and the ATCC.
Reaction Mixture for Real Time-PCR on LightCycler 480 (ROCHE):
10 μl RT HS-PCR Mix SYBR® A (A&A Biotechnology)
8 μl H2O sterile water, nucleases-free. Treated with DEPC. (A&A Biotechnology)
1 μl Primer mix (solution in 10 mM Tris-HCl pH 8.0) (2.5 μM each)
1 μl DNA (in negative control—1 μl of sterile water)
PCR Reaction Conditions:
Initial denaturation: 95° C. 5 min
45 cycles:
Step 1—95° C. 15 s
Step 2—55° C. 25 s
Step 3—72° C. 15 s
Melting—65-97° C.
Combining the results of the growth curves and the melting curves, it is apparent that for group 1 the expected hybridization and subsequent amplification between the inventive primers, in particular inventive primer pair and the chloramphenicol acetyltransferase target sequence took place, whereas for groups 2 and 3 the respective growth and melting curves are associated with the creation of primer-dimers not resulting in the expected amplification of the of the chloramphenicol acetyltransferase target sequence. Table 4 provides an overview of the respective executive examples 1 and 2.
C. difficile
Table 4 shows that the results of the PCR study of executive example 2 approves the in silico results of executive example 1, which means that the inventive primer pair is highly specific for the detection of hyper-virulent Clostridioides difficile strain of the B1/NAP1/027 group in comparison to closely related strains of group 2 or other strains of group 3. The PCR amplification method is also highly sensitive as well as simple, time and cost effective.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19209383.9 | Nov 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/082206 | 11/16/2020 | WO |
