Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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Recruitment of cyanobacteria by reverse transcription quantitative real-time PCR based on expression of Microcystis gene

PeerJ ◽

10.7717/peerj.7188 ◽

2019 ◽

Vol 7 ◽

pp. e7188

Author(s):

Long Yu ◽

Xiaofei Wu ◽

Yang Yu ◽

Limei Shi ◽

Min Zhang

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lake Taihu ◽

Rna Extraction ◽

Sybr Green ◽

Quantitative Detection ◽

Control Group ◽

Quantitative Real Time Pcr ◽

Total Rna ◽

Pcr Method

In this study, a SYBR Green quantitative real-time PCR method was established and applied. Relative expression of the synthetic genes from Microcystis gas vesicles (gvpC), algal toxin genes (mcyA), and polysaccharides (espL) from water and sediments of Meiliang Bay and from the center of Lake Taihu were tested from January to June, 2017. Indoor Microcystis aeruginosa was used as the control group. The kit for total RNA extraction in Microcystis was optimized. Results showed that the optimized kit extracted high-concentrations and high-quality total RNA from Microcystis. The extraction purity and concentration were significantly higher than those extracted by the original kit. The transcription level of gvpC increased gradually until a peak was reached in March. However, expression of gvpC decreased continuously at the proliferating and floating stages of Cyanobacterial biomass. The maximum level of expression of gvpC in April in comparison to expression of mcyA in March occurred first. We found that the SYBR Green qRT-PCR method, which is characterized by high specificity, repeatability, is rapid, and can be used for quantitative detection of expression of gvpC, mcyA, and espL. The recruitment of cyanobacteria is the process in which cyanobacteria in the sediment began to regain their activity, started to grow and migrated to the water column.

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Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3911-3916.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3911-3916 ◽

Cited By ~ 71

Author(s):

Mark G. Wise ◽

Gregory R. Siragusa

Keyword(s):

Gastrointestinal Tract ◽

Real Time ◽

Clostridium Perfringens ◽

Real Time Pcr ◽

Limit Of Detection ◽

Quantitative Detection ◽

Necrotic Enteritis ◽

Analytical Sensitivity ◽

Quantitative Real Time Pcr ◽

Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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Quantitative real-time PCR method to detect changes in specific transcript and total RNA amounts

Electronic Journal of Biotechnology ◽

10.2225/vol7-issue1-fulltext-5 ◽

2004 ◽

Vol 7 (1) ◽

Author(s):

Daniel Z. Skinner ◽

Kwang-Hyun Baek

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Total Rna ◽

Specific Transcript ◽

Pcr Method

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Collaborative Validation of an Event-Specific Quantitative Real-Time PCR Method for Genetically Modified Rice Event TT51-1 Detection

Journal of Agricultural and Food Chemistry ◽

10.1021/jf401339k ◽

2013 ◽

Vol 61 (25) ◽

pp. 5953-5960 ◽

Cited By ~ 14

Author(s):

Yuhua Wu ◽

Litao Yang ◽

Yinglong Cao ◽

Guiwen Song ◽

Ping Shen ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genetically Modified ◽

Quantitative Real Time Pcr ◽

Genetically Modified Rice ◽

Pcr Method

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A Quantitative Real-Time PCR Method Using an X-Linked Gene for Sex Typing in Pigs

Molecular Biotechnology ◽

10.1007/s12033-012-9589-5 ◽

2012 ◽

Vol 54 (2) ◽

pp. 493-496 ◽

Cited By ~ 5

Author(s):

Maria Ballester ◽

Anna Castelló ◽

Yuliaxis Ramayo-Caldas ◽

Josep M. Folch

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Sex Typing ◽

Linked Gene ◽

Pcr Method

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Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

Archives of Pathology & Laboratory Medicine ◽

10.5858/134.3.444 ◽

2010 ◽

Vol 134 (3) ◽

pp. 444-448 ◽

Cited By ~ 1

Author(s):

Zhengming Gu ◽

Jianmin Pan ◽

Matthew J. Bankowski ◽

Randall T. Hayden

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Bk Virus ◽

Clinical Samples ◽

Quantitative Detection ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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Detection of Rendered Meat and Bone Meals by PCR Is Dependent on Animal Species of Origin and DNA Extraction Method

Journal of Food Protection ◽

10.4315/0362-028x-73.6.1090 ◽

2010 ◽

Vol 73 (6) ◽

pp. 1090-1096 ◽

Cited By ~ 9

Author(s):

MICHAEL J. MYERS ◽

DOROTHY E. FARRELL ◽

CHRISTINE M. DEAVER ◽

JACQULINE MASON ◽

HEIDI L. SWAIM ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Animal Species ◽

Animal Feed ◽

Functional Assay ◽

Threshold Values ◽

Meat And Bone Meal ◽

Bone Meal ◽

Pcr Method

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

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Multiplex Quantitative Real-Time PCR for the Detection of t(14;18) Translocations with Breakpoints within 5 Different Regions of the BCL-2 Gene: MBR, 3′MBR, mcr, 5′mcr, icr.

Blood ◽

10.1182/blood.v106.11.2826.2826 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2826-2826

Author(s):

Frank Schüler ◽

Sandra C. Dölken ◽

Carsten Hirt ◽

Gottfried Dolken

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Mononuclear Cells ◽

Consensus Sequence ◽

Degraded Dna ◽

Quantitative Detection ◽

Target Sequence ◽

Quantitative Real Time Pcr ◽

Consensus Primer ◽

Target Sequences

Abstract Follicular lymphomas (FL) are associated with the chromosomal translocation t(14;18)(q32;q21). Most breakpoints of chromosome 18 (60%) occur in the major breakpoint region (MBR) of the BCL-2 gene. Further breakpoints have been detected in the minor cluster region (mcr), less frequent breakpoints are found in regions called 3′-MBR, 5′-mcr and icr. On chromosome 14 most breakpoints are located within one of the six JH-genes. Therefore, BCL-2 translocations with breakpoints within the MBR and mcr are generally detected by PCR using combinations of different BCL-2 primers with one JH-consensus primer. We have developed a multiplex quantitative real-time PCR strategy that that can be used to detect t(14;18) translocations with breakpoints located within all regions mentioned above. To minimize the costs for expensive probes we used the JH-consensus sequence as a target for one “consensus probe” (fluorescent labelled minor groove binder probe) for all assays in combination with 6 different JH intron primers. To reduce the size of amplified PCR fragments 12 BCL-2 primers were chosen in combination with 6 JH intron primers for the detection of all 5 breakpoint regions. It is very important to choose short DNA target sequences for amplification: (a) to establish a real-time PCR with a high amplification efficacy; (b) to be able to amplify target sequences also from partially degraded DNA isolated from formaldehyde-fixed paraffin-embedded tissue sections; (c) to achieve a high sensitivity to detect 1–3 copies per assay. Peripheral bood mononuclear cells (PBMNC) and formalin fixed, paraffin embedded lymph node tissue obtained from 139 FL patients at the time of diagnosis (LN and PBMNC, n = 54; LN only, n = 3; PBMNC only, n = 82) were tested by multiplex quantitative real-time PCR. 80 breakpoints were identified within the MBR (61%) region. For comparison, 78/80 breakpoints were also detected by our standard real-time PCR assay with one BCL-2-MBR- primer and one JH consensus primer in combination with a fluorescent probe located within the BCL-2 sequence [Doelken et al., BioTechniques, 1998]. Two additional translocations with breakpoints located 5′ of the target sequence of the standard PCR were found by using two additional MBR primers. In addition, five mcr breakpoints (5%), one breakpoint in the 3′MBR region and one breakpoint in the icr region were found. Based on these results the prevalence of breakpoints in various regions of the BCL-2 gene in FL patients is: MBR = 61% (80/139); mcr = 5% (5/139); 3′MBR = 1% (1/139); icr = 1% (1/139); 5′mcr = 0%). Furthermore, based on quantitative PCR results the t(14;18) translocations detected in this study were undoubtedly lymphoma associated and did not belong to t(14;18)-positive non-lymphoma B cell clones found in healthy persons. By applying this multiplex quantitative real-time PCR strategy t(14;18) translocations with breakpoints in five different breakpoint clusters can be detected in about 70% of patients with follicular lymphoma. The assays can be used for a fast and reliable quantitative detection of t(14;18) translocations on DNA isolated from fresh lymph nodes or pathological specimens as well as blood samples at the time of diagnosis. In almost all cases quantitative results will allow a distinction whether the translocation found is lymphoma associated or not, which will in turn allow a quantitative MRD analysis on follow-up samples during and after treatment.

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