scholarly journals Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment

2021 ◽  
Vol 11 ◽  
Author(s):  
Shuo Zhao ◽  
Jingyun Zhang ◽  
Zhe Li ◽  
Yu Han ◽  
Biao Kan
Keyword(s):  
Vibrio Cholerae ◽  
Quantitative Pcr ◽  
Bacterial Species ◽  
Accurate Method ◽  
Single Copy ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Vbnc State

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

scholarly journals Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

Water ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3507
Author(s):  
Mark A. Ibekwe ◽  
Shelton E. Murinda ◽  
Stanley Park ◽  
Amarachukwu Obayiuwana ◽  
Marcia A. Murry ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Point Of Care ◽  
Foodborne Pathogen ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
High Rate ◽  
E Coli

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

A Microbiological Paradox: Viable but Nonculturable Bacteria with Special Reference to Vibrio cholerae

1996 ◽  
Vol 59 (1) ◽  
pp. 96-101 ◽  
Author(s):  
ANWARUL HUQ ◽  
RITA R. COLWELL
Keyword(s):  
Vibrio Cholerae ◽  
Culture Media ◽  
Direct Detection ◽  
Bacterial Species ◽  
Detection Methods ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Bacteriological Culture ◽  
Nonculturable State

The observation that directly-detectable bacterial cells are unable to grow on bacteriological culture media under certain conditions raises questions regarding the viability of these cells. Various terminologies have been used to describe substrate-responsive and metabolically-active bacterial cells that cannot be cultured. The currently-accepted term is “viable but nonculturable.” During the past 15 years, the viable but nonculturable phenomenon has been actively investigated. Bacterial pathogens in the viable but nonculturable state can maintain virulence and produce disease. These organisms may escape detection if bacteriological culture methods are solely used. Thus, methods for direct detection of specific pathogens in food, water and environmental samples are preferable. Viable but nonculturable Vibrio cholerae have been extensively studied, and several sensitive and reliable direct-detection kits have been developed. Viable but nonculturable forms of bacteria are now recognized as a common phenomenon, observable in many bacterial species, which suggests that standard bacteriological laboratory protocols for assessing microbiological safety of food and drinking water are less reliable than direct detection methods.

PSX-B-15 Real-time quantitative PCR versus droplet digital PCR for measurement of peripheral blood leukocytes interferon-tau stimulated genes in beef cattle

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 326-326
Author(s):  
Gabriela Dalmaso de Melo ◽  
Gessica A Franco-Johannsen ◽  
Brooke McAnnally ◽  
Reinaldo F Cooke ◽  
Ky G Pohler
Keyword(s):  
Quantitative Pcr ◽  
Reference Gene ◽  
Peripheral Blood ◽  
Digital Pcr ◽  
Beef Cows ◽  
Droplet Digital Pcr ◽  
Peripheral Blood Leukocytes ◽  
Interferon Tau ◽  
Blood Leukocytes ◽  
Real Time Quantitative Pcr

Abstract We aimed to compare the abundance of interferon-tau stimulated genes (ISG) transcripts in peripheral blood leukocytes of artificially inseminated beef cows using real-time quantitative PCR (RT-qPCR) versus Droplet Digital PCR (ddPCR). Multiparous Bos taurus beef cows (n = 7) were subjected to timed artificial insemination (TAI) on day 0. Pregnancy was determined by transrectal ultrasonography on days 26 and 30 post-TAI, and cows were classified as: pregnant (n=4; embryo detected on days 26 and 30) or non-pregnant (n = 3; no embryo detected). Coccygeal vein blood samples were collected on days 0, 15, 17, 19, 20 and 24 post-TAI. Leukocyte RNA was extracted from the buffy coat fraction using Trizol associated with the DirectZol-RNA kit and transcribed to cDNA. The abundance of ISG (ISG15 and MX2) was assessed by relative quantification to a reference gene (RPS9) using RT-qPCR and by absolute quantification using the QX100TM Droplet DigitalTM PCR System (Bio-rad Laboratories) according to manufacturer’s recommendations. Data were analyzed using PROC MIXED on SAS 9.4. For the RT-qPCR, pregnant cows had greater (P &lt; 0.05) ISG15 and MX2 abundance compared to non-pregnant cows on days 20 (ISG15:0.11±0.1 vs. 0.01±0.001 and MX2:0.73±0.4 vs. 0.06±0.06) and 24 (ISG15:0.34±0.2 vs. 0.01±0.001 and MX2:0.77±0.2 vs. 0.13±0.04). For ddPCR, a greater ISG15 and MX2 copy numbers in pregnant cows was observed on days 15 (ISG15:129 vs. 44 copies/µl and MX2:33 vs. 10 copies/µl) and 20 (ISG15: 216 vs. 30 copies/µl and MX2: 44 vs. 7 copies/µl), and also on day 24 for ISG15 (32 vs. 7 copies/µl) compared to non-pregnant cows. In conclusion, ddPCR was able to detect an earlier expression of ISG in pregnant cows. Future studies are needed to enroll more animals and establish a suitable cutoff value using ddPCR, which could be less subjective for diagnosis as it does not require the use of a reference gene.

Improved Igh-Based MRD Detection By Using Droplet Digital PCR: a Comparison With Real Time Quantitative PCR In MCL and MM

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4290-4290
Author(s):  
Daniela Drandi ◽  
Lenka Kubiczkovà ◽  
Nadia Dani ◽  
Simone Ferrero ◽  
Luigia Monitillo ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Research Funding ◽  
Residual Disease ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Chain Gene ◽  
Attractive Alternative ◽  
Standard Curve ◽  
Real Time Quantitative Pcr

Abstract Background In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above. Methods Bone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results Overall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p<0.0001) (fig). DD-PCR and RQ-PCR showed superimposable sensitivity (10-5). Specificity in terms of appearance of non-specific amplifications signals in no-template samples (tested for all patients) and reproducibility on 30 replicates (4 samples) were superimposable. 128 out of 161 samples were fully concordant (Choen's K=0.80). MRD detection was concordantly positive in 106/161 (65.8%) samples and concordantly negative in 22/161 samples (13.7%). Only 5/161 (3.1%) samples showed major qualitative discordance. 28/161 (17.4%) samples showed minor qualitative discordance (which might be related to Poisson's statistics). Quantitative discordances were observed in 5/161 (3.1%) of cases (positive non quantifiable (PNQ) cases were conventionally placed to a value intermediate between sensitivity and quantitative range). Interestingly, 17 samples negative by RQ-PCR were scored positive by DD-PCR (median 6 copies, range 2-74) while 16 samples positive by RQ-PCR (median 5 copies, range 2-44) were negative by DD-PCR. Conclusions Here we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment. Disclosures: Kubiczkovà: GAP304/10/1395 : Research Funding; MUNI/11/InGA17/2012: Research Funding.

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