Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding.
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ABSTRACT:
SUBMITTER: Takasaki K
PROVIDER: S-EPMC8104373 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices.
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Project description:The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.
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Project description:Real-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as the qnrA, qnrB, and qnrS genes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification of qnr genes in complex samples.
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Project description:Adult female mice (C57BL/6) were super-ovulated by intraperitoneal hormone injections with the interval of 48 hours between injections. The hormones administered were PMSG followed by hCG (5IU/0.1ml, Sigma-Aldrich). Following the second hormonal treatment, the females were mated with males (1:1, C57BL/6). Zygotes, 2-cell and 4-cell and blastocysts were collected 26, 48 and 52 hours post hCG. Prior to cell collection, the zona pelucida was removed by Tyrode s acid solution. The 2- and 4-cell embryos were then immersed in Trypsin solution (Invitrogen) with RNase inhibitor (1 IU/ul, Clontech) and BSA (50 ug/ul) for the separation of the blastomeres. The separated blastomeres were snap frozen, and maintained at -80 C until processed. Twelve zygotes, 20 2-cell and 9 4-cell embryos were collected for single cell qPCR. In addition, we prepared with a pool of 10 zygotes and a pool of 50 zygotes as positive controls. The cells and pools were subjected to reverse transcription with SuperScript VILO cDNA Synthesis kit (Life Technologies) as recommended by the manufacturer. DELTAgene Assays were custom designed for 94 genes associated with embryonic development or regulation of gene expression, as well as 2 housekeeping genes. The cDNA from cells and pools were subjected to target specific amplification for 20 cycles. Pre-amplified template was diluted and subjected to primer specific amplification with SooFast EvaGreen Supermix (Biorad) on 96.96 array chip and BiomarkHD System (Fluidigm). Relative quantification of gene expression (log2 space) was obtained by subtracting the Ct values from the baseline value of 22.
2014-09-01 | GSE59892 | GEO
Project description:Adult female mice (C57BL/6) were super-ovulated by intraperitoneal hormone injections with the interval of 48 hours between injections. The hormones administered were PMSG followed by hCG (5IU/0.1ml, Sigma-Aldrich). Following the second hormonal treatment, the females were mated with males (1:1, C57BL/6). Zygotes, 2-cell and 4-cell and blastocysts were collected 26, 48 and 52 hours post hCG. Prior to cell collection, the zona pelucida was removed by Tyrode s acid solution. The 2- and 4-cell embryos were then immersed in Trypsin solution (Invitrogen) with RNase inhibitor (1 IU/ul, Clontech) and BSA (50 ug/ul) for the separation of the blastomeres. The separated blastomeres were snap frozen, and maintained at -80 C until processed. Twelve zygotes, 20 2-cell and 9 4-cell embryos were collected for single cell qPCR. In addition, we prepared with a pool of 10 zygotes and a pool of 50 zygotes as positive controls. The cells and pools were subjected to reverse transcription with SuperScript VILO cDNA Synthesis kit (Life Technologies) as recommended by the manufacturer. DELTAgene Assays were custom designed for 94 genes associated with embryonic development or regulation of gene expression, as well as 2 housekeeping genes. The cDNA from cells and pools were subjected to target specific amplification for 20 cycles. Pre-amplified template was diluted and subjected to primer specific amplification with SooFast EvaGreen Supermix (Biorad) on 96.96 array chip and BiomarkHD System (Fluidigm). Relative quantification of gene expression (log2 space) was obtained by subtracting the Ct values from the baseline value of 22. 12 zygotes, 10 2-cell, and 9 4-cell mouse (C57BL/6) embryos were collected and multi-cell embryos were separated into blastomeres
2014-09-01 | E-GEOD-59892 | biostudies-arrayexpress
Project description:Several parasite species are shared between humans and pigs. We explored the application of next-generation sequencing-based metabarcoding supplemented with real-time PCR to fecal DNAs from 259 samples from 116 pigs in Denmark to detect and differentiate single-celled intestinal parasites of zoonotic relevance. Enterocytozoon bieneusi, Balantioides coli, and Giardia duodenalis were observed in 34/37 (92%), 148/259 (57%), and 86/259 (33%) samples, respectively. Entamoeba polecki ST1, E. polecki ST3, and Entamoeba hartmanni were detected in 104/259 (40%), 161/259 (62%), and 8/259 (3%) samples, respectively. Metabarcoding and real-time PCR detected Cryptosporidium in 90/259 (35%) and 239/259 (92%) of the samples, respectively, with Cryptosporidium suis and Cryptosporidium scrofarum observed in nearly equal proportions. Blastocystis subtypes 1, 3, 5, and 15 were found in 72 (28%), 6 (2%), 176 (68%), and 36 (14%) of 259 samples, respectively. Iodamoeba was identified in 1/259 samples (<1%), while none of 37 tested samples was positive for Dientamoeba fragilis. Our results illustrate how metabarcoding exemplifies a 'one-fits-many' approach to detecting intestinal single-celled parasites in feces supplemented with real-time PCR for selected parasites. Using metabarcoding with pathogen-specific assays may help detect emerging and previously underdetected pathogens and further elucidate the role of micro-eukaryotic parasites in human and animal health and disease.
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Project description:The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.
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Project description:Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.
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Project description:An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.
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Project description:The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method.IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.
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