Patent Application
20200095624
The present invention relates to methods and reagents for assaying a sample for the presence of fecal contamination from humans.
Much human disease has been transmitted via fecal contaminated water. Often disease-causing bacteria and viruses found in feces are the causative agent. Modern sewage treatment is primarily focused on killing or removing of these pathogenic microbes prior to discharge into the environment.
Testing for the presence of indicator bacteria typically found in and transmitted in feces is a well-established approach and is presumptive of fecal contamination in a water sample. The water, food, objects and environmental samples so contaminated are presumably unsafe for human consumption and contact.
Most current health, safety and regulatory methods used to assess water and food quality rely on measuring the levels of culturable indicator bacteria of fecal contamination, such as enterococci and fecal coliforms. These act as a proxy for pathogenic viruses potentially present in feces as well. However, these general fecal indicator methods do not discriminate among different bacterial strains which are found in human fecal contamination as opposed to other animal sources of fecal contamination. While these animal strains may be pathogenic as well, the presence of human strains typically represent a greater public health risk. Also, knowing the source for the strain allows one to identify the source of the contamination by microbial source tracking (MST), and to remedy the situation, such as repairing a faulty sewage treatment plant.
Other approaches have been attempted to determine sources of fecal contamination in the environment. One technique is a PCR-based method that identifies human fecal pollution by targeting bacterial 16S rRNA gene sequences from Bacteroides (Bernard and Field, AEM 66:4571-4574, 2000). However, this approach targets bacteria rather than virus microorganisms. The present invention was developed to produce a fast, sensitive and specific assay for human fecal contamination utilizing a viral indicator.
Previously, applicants have developed assays to distinguish bacterial strains from various animal species for fecal contamination detection as a tool for MST. For example, U.S. Pat. Nos. 8,574,839, 8,058,000 and 7,572,584.
The Cross-Assembly phage (CrAssphage) was first described by Dutilh, B. E., Cassman, N., McNair, K., Sanchez, S. E., Silva, G. G., Boling, L., & Edwards, R. A. (2014). A highly abundant bacteriophage discovered in the unknown sequences of human fecal metagenomes. Nature communications, 5 as an approximately 97 kbp double stranded DNA circular genome discovered by assembly of sequence reads from a human fecal metagenome. Since the genome was derived from the metagenomics reads, the genome represents a consensus genome of viral quasispecies. They report 80 predicted protein coding genes, two-thirds of which had no predicted function, demonstrating why the phage has not been previously discovered. Co-occurrence profiling predicted a bacterial Bacteroides host. They also reported that the genome was most prevalent in human fecal samples and sewage.
Based on the information in this paper, an initial metagenome evaluation was completed in Stachler, E., & Bibby, K. (2014). Metagenomic evaluation of the highly abundant human gut bacteriophage CrAssphage for source tracking of human fecal pollution. Environmental Science & Technology Letters, 1(10), 405-409. In this preliminary study, 86 metagenomes from different environments were evaluated for the presence of CrAssphage by mapping metagenomic reads against the consensus genome. The CrAssphage genome was found to be abundant in sewage samples from the U. S. and Europe while being less abundant in sewage samples from Africa and Asia. In addition, crAssphage was found to be relatively absent in samples from other animals, with the exception of one bat guano sample. Upon further inspection it was found that nearly half of the reads mapping from the bat metagenome mapped to a single open reading frame (ORF) of the crAssphage genome. In addition, sewage metagenome reads from the U. S. and Europe were mapped against other viral genomes previously suggested as human-associated fecal source identification genetic markers, showing that crAssphage is significantly more abundant in the metagenomics reads than the other known viruses.
Despite the initial screening of the crAssphage genome, many challenges remain in creating a human-associated genetic marker for fecal source identification applications. First, since the crAssphage genome was developed from one individual and represents a consensus sequence, it may include errors that lead to unsuitable genetic markers. In addition, there is currently no laboratory data confirming the animal host range, geographic stability, or detection in an environmental sample. Furthermore, with little more than theoretical data, the sensitivity and specificity of crAssphage in actual environmental samples is not determined in a manner as to determine whether one can actually develop and assay.
It is an object of the present invention to assay for the human specific region of crAssphage. It is a further object of the present invention to provide methods for identifying whether microbial containing sample is from a human fecal-contaminated material. It is still another object of the present invention to provide DNA primers or probes which can specifically hybridize to and allow determination of the presence of the humanassociated-region of crAssphage.
The present invention performs the assay functions for two regions of crAssphage that are strongly associated for human sourced crAssphage. The presence of the sequences in these two regions may be determined by a variety of standard molecular biology techniques on crAssphage containing samples.
The crAssphage of the present invention has been described previously. The listed sequence maybe a metagenomic sequence derived from databases of human fecal DNA. The sequence numbering in the crAssphage genome is a composite and the sequence numbering may vary slightly with sequenced true biological samples. Thus, the sequence numbering is provided for convenience and ease of understanding rather than a definition. Also, crAssphage appears to be a bacteriophage and is discussed herein as if it is due to its association with Bacteroides. However, this has yet to be proved conclusively and the term should be viewed as a convenience for ease of understanding only.
“Human specific region” refers to sequences within crAssphage that are found in crAssphage that are associated with human fecal bacteria, but not other animal versions of crAssphage. Slight overlap with unusual animal sources of fecal contamination, such as the odd result with certain bat fecal samples, are still considered “human specific” for the purposes of the present invention.
The present invention describes several potentially human related regions provided in the Table 3 below. More preferred are laboratory confirmed human related regions of crAssphage listed in Tables 4 and 5. Human specific regions of particular interest are the regions containing any part of the genomic region of crAssphage amplified by respective PCR primers. The human specific region may encompass the entire amplified region between and including the primers or it may include regions outside these sequences provided that it at least partially overlaps the region defined by the PCR primers described above and below.
Alternatively, the human specific region maybe defined based on amplified regions which are NOT human specific. In the examples below, a large number of PCR amplified regions were tested. While many were not human specific, untested regions between the tested regions may also be human specific. Of particular interest are untested regions adjacent to suspected human specific regions. Regardless, any region determined as not human specific by the methods in the examples are considered not human specific.
Reagents used in the assay of the present invention include primers, probes, and/or other oligonucleotides. These may be directly labeled, indirectly labeled or labeled and/or stained after hybridization. Many nucleotide labeling techniques are known per se. Likewise, a number of suitable labeling techniques are well known per se, such as fluorescent, quenching, enzyme, ligand, etc. The label may indicate either the presence or absence of hybridization of the primer(s) or probe(s), as long as it is sufficiently detectable to answer the question of whether the human specific region(s) of crAssphage are present in a sample. Techniques for using such a primer or probe in a variety of different assay formats are well known per se. For example, in the examples below, both end-point PCR and qPCR have been used. A real-time PCR, dPCR, ddPCR, RT-PCR and other known techniques may be used also. Primer extension products may be determined by unrelated techniques such as by mass spectrometry. Further examples include long probes to the human-specific region wherein the probe(s) may be labeled before or after hybridization. In short, any standard molecular biology method for detecting a particular sequence may be used in the present invention to detect the presence or absence of human specific region(s) in crAssphage.
Hybridization or annealing of a primer or probe is preferably completely complementary. However, a slight non-complementarity approach may be tolerable to account for random mutations and sequence variations in different human crAssphage genomes.
CrAssphage is very abundant in fecal samples both in quantity and in its widespread locations around the world. Thus, most conventional nucleotide-based assays should not have an issue with sensitivity. An extremely dilute sample may offer challenges with sensitivity, but that is a sampling issue rather than an assay problem.
The sample suspected of containing human crAssphage may be from any natural or artificial source that could possibly be exposed to feces. Examples include, environment samples of water, soil, air, ground water, vegetation, the surface wild or domesticated animals. Artificial sources include waste water, surface run-off, aerosols, food products, feed products, fiber products, manufactured goods of all kinds, but especially pharmaceuticals, medical devices, medicaments, door knobs, handrails, and anything else that can contact a human body. The sample maybe from a person (external or internal) or laboratory sample such as a culture.
This data suggests that crAssphage could be a highly specific and sensitive human fecal source identification indicator. Several regions of the crAssphage genome were identified by manual inspection to be highly abundant across human metagenome samples. These regions were searched for crAssphage specific primers using PrimerBLAST and several regions were reported for being ideal for technology development (crAssphage genome bp positions: 1770-1870, 78100-78270, 83860-83970, 88370-88470, 90120-90280, and 93160-93340).
In order to identify candidate crAssphage primer sets, suitable for a human-specific fecal source identification technology, a unique multistep strategy was employed (
After identification of probable human-specific genetic regions and the development of candidate primer sets, three stages of end-point PCR testing were conducted to identify the most suitable human-specific crAssphage fecal source identification technology (Phase II, III, and IV). The details for each Phase are detailed below.
Candidate primer sets that pass all end-point PCR screens were then adapted to a qPCR technology in order to develop a quantitative technology appropriate for human MST applications (Phase V).
The following non-limiting example is provided to illustrate the present invention.
The crAssphage metapopulation consensus genome of viral quasispecies was used as a template for candidate primer design to develop a human-specific fecal source identification technology. PrimerBLAST was used to design candidate primers with default parameters except product length was restricted to a range of 90 and 180 bps. When multiple primer pairs were suggested for a particular region, primer selection was based on optimizing the 3′ end specificity, including 2-3 C or G for the GC clamp, looking for primers with higher Tm and similar Tm within the pair, higher GC content, and eliminating self-complementarity. Eligible genetic regions for candidate end-point PCR primer design were selected based on a predefined set of criteria including:
In total, 384 candidate primer sets were designed targeting the crAssphage metapopulation consensus genome of viral quasispecies. All candidate primer sets are available upon request. During selection, 45,940 bp were found to be eligible for primer design. The 384 primer pairs and their products represent 41,794 bp, representing 91% coverage of the eligible region.
Round 1 was designed to identify candidate primer sets that exclusively amplify human sewage without eliciting false positive detections to select non-target animals. Testing was conducted using two composites including (1) raw sewage and (2) non-target animals (pig, cow, dog, and goose).
Composite DNA samples were made to test the primer pairs in the first round of testing. Sewage samples were collected from three different sites in Cincinnati, OH. DNA was extracted using the QIAamp DNA Blood Maxi Kit substituting Buffer AVL for Buffer AL. The samples were pooled and the composite was diluted to 0.5 ng/μL for a total of 1 ng/reaction. For the non-target animal composite, DNA was extracted from animal fecal samples using a modified procedure of the GeneRite DNA-EZ Kit. Nine individual samples were used for each of the four animal groups including pig, cow, dog, and goose. Samples were pooled and the composite was diluted to 2 ng/μL for a total of 4 ng/reaction (1 ng/reaction of DNA from each animal group). Each candidate primer pair was subjected to six reactions, duplicates each of the sewage composite (1 ng/reaction), the non-target animal composite (4ng/reaction), and no template controls.
Amplification conditions were PCR screening are described in Table 1. All end-point PCR reactions were run on a Tetrad 2 thermal cycler (BioRad Laboratories) under the following conditions: 94° C. for 5 min and 40 cycles of 40 s at 94° C., 1 min at 57° C., and 30 s at 72° C.
PCR products were visualized by electrophoresis on 2.0% lithium borate buffer gels using a UVP gel imager. Refer to
Example gel from Round 1 (
Results of Round 1 screening are listed in Table 2. In summary, only 57 candidate primer sets were eligible for Round 2 testing (complete data set available upon request). Of the 384 primers, 31.5% failed to detect the sewage composite. This included a large region of the genome where no primers worked (crAssphage genome locus 25607 to 43723 bp) suggesting that this region may be present at too low of a concentration to detect, represented a region of genetic variation between different quasispecies, or indicates errors in the reported crAssphage consensus genome. Regardless, data indicates that this region is not suitable for human fecal source identification technology development. In addition, 6.8% of primers tested showed false positives, 2.6% had spurious bands in sewage composite, and 1.3% had spurious bands in non-target animal composite, eliminating them from further testing. Of all the primer sets tested, only one had a positive NTC. The rest of the primers were eliminated from further testing due to presence of undesirable primer dimerization amplification products.
Round 2 was designed to test candidate primer set sensitivity to sewage and increase test concentrations of non-target animals to more rigorously assess specificity. For sensitivity testing, three dilutions were prepared from the sewage composite used in Round 1 including test concentrations of 0.1 ng/reaction, 0.01 ng/reaction, and 0.001 ng/reaction. For specificity testing, each non-target animal group was tested individually at a test concentration of 5 ng/reaction. The same reaction composition and thermal cycling conditions for Round 1 were used.
PCR products were visualized by electrophoresis on 2.0% lithium borate buffer gels using a UVP gel imager. Refer to
In total, six candidate primer sets passed all selection criteria and were deemed eligible for
Round 3 testing.
Example gel from Round 2 Testing. Pictured in
Round 3 represents the most rigorous level of testing designed to select the top performing candidate primer sets for specificity, sewage geographic distribution in the United States, environmental detection demonstration, and PCR product sequencing. The six candidate primer sets that passed Round 2 were tested (Table 3).
Specificity. Excellent specificity is the foundation of any useful microbial source tracking technology. Candidate primer sets passing Round 2 were tested against a panel of non-target animal sources. Fecal reference samples include domestic dog, pig, cattle, Canada goose, whitetail deer, horse, elk, duck, beaver, and gull. Each animal group will consist of 9 individual samples. Each sample was tested in triplicate at a 1 ng/reaction test concentration (total of 270 reactions per primer set). Resulting data was used to calculate specificity [true negatives/(true negatives+false positives)] for each candidate primer set.
U.S. Sewage Geographic Distribution. Computer analyses of United States sewage sample metagenomic libraries suggests that the crAssphage is highly abundant. Candidate primer sets passing Round 2 were tested against raw sewage samples collected from 10 different geographic locations across the United States. Each sewage preparation was tested in triplicate at 1 ng/reaction
Limit of Detection (LOD95). The limit of detection of each candidate primer set passing Round 2 was tested to characterize the lowest sewage template concentration detected in 95% of replicate samples. A composite of DNA sewage samples from 10 different geographic locations was tested at five concentrations ranging from 1 ng/reaction to 0.0001 ng/reaction. For each test concentration, 20 replicates were performed to calculate the proportion of positives. The lowest test concentration where at least 95% of replicates were positive was defined as the LOD95.
Environmental Detection Demonstration. The ultimate goal for a crAssphage human-specific microbial source tracking technology is to detect human pollution in unknown samples. Even though a particular candidate primer set may yield a detectable PCR product in a sewage sample, the genetic target may not persist in the sample environment at detectable concentrations. To demonstrate detection in an environmental sample, each candidate primer set passing Round 2 was tested against a sewage impaired water sample collected from a local stream.
Top performing crAssphage genomic regions for human fecal pollution identification are listed in Table 4. Additional data will be available in future publication.
The top two performing primer sets based on results from Round 3 testing were adapted to the TaqMan qPCR technology. A BLASTn search using the nr database identified crAssphage056 and craAssphage064 sequences encoding for hypothetical proteins of the crAssphage genome Orf000024 and 0000025, respectively. Genomic regions include:
Primers and hydrolysis probes were designed using Life Technologies Primer Express Software and expert judgement (Table 5).
In addition, a customized DNA standard was developed for calibration model generation. qPCR technologies were evaluated for calibration model performance, abundance in target and non-target samples, as well as performance in environmental water samples. Calibration model performance of qPCR assays is shown in Table 6.
Specificity and sensitivity testing were conducted with 222 individual fecal and sewage samples collected from 10 different geographic locations across the United States. Table 7 summarizes results for both end-point and qPCR crAssphage0056 and crAssphage064 assays. Specificity and sensitivity test reactions were standardized to 1 ng/reaction of total DNA. For qPCR data, only results above the lower limit of quantification (LLOQ) were scored as false positives.
To characterize the abundance of crAssphage056 and crAssphage064 human-specific genetic markers in common pollution sources, each assay was tested against a collection of 224 sewage, fecal, and environmental water samples collected from 10 different geographic locations across the United States. Table 8 summarizes the range of concentrations observed in log10 copies/reaction. For fecal and sewage samples, test reactions were standardized to 1 ng/reaction of total DNA.
The quality of findings was verified through a series of rigorous controls. The absence of contamination was confirmed in both no template control (n=112) and extraction blank reactions (n=27). For environmental water samples, a sample processing control was included with each DNA extract. All sample processing controls demonstrated the absence of matrix interference. Amplification inhibition for all DNA extracts was monitored with internal amplification controls using HF183/BacR287 and HumM2 qPCR multiplex assays. Only 98.7% of all DNA extracts exhibited no inhibition. DNA extract with amplification inhibition (cow=2; gull=1) were discarded from the study.
It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.
All patents and references cited herein are explicitly incorporated by reference in their entirety.
The present application claims priority from provisional application Ser. No. 62/386,532, filed Dec. 4, 2015, the entire contents of all of which are hereby incorporated by reference. The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 7, 2019, is named 0075_1014_SL.TXT and is 1,155 bytes in size.
This invention was made with government support from the Environmental Protection Agency. The United States government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62386532 | Dec 2015 | US |
