Real-time PCR assay may be used to verify suspicious test results of Ureaplasmas spp. from the liquid culture method

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

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A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

Journal of Food Protection ◽

10.4315/0362-028x-70.5.1080 ◽

2007 ◽

Vol 70 (5) ◽

pp. 1080-1087 ◽

Cited By ~ 63

Author(s):

V. M. BOHAYCHUK ◽

G. E. GENSLER ◽

M. E. McFALL ◽

R. K. KING ◽

D. G. RENTER

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Pcr Assay ◽

Culture Methods ◽

The Real ◽

Conventional Culture ◽

Food Animal ◽

Highly Sensitive ◽

Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

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Performance and Verification of a Real-Time PCR Assay Targeting thegyrAGene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae: TABLE 1

Journal of Clinical Microbiology ◽

10.1128/jcm.03032-15 ◽

2016 ◽

Vol 54 (3) ◽

pp. 805-808 ◽

Cited By ~ 34

Author(s):

P. Hemarajata ◽

S. Yang ◽

O. O. Soge ◽

R. M. Humphries ◽

J. D. Klausner

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Real Time Pcr ◽

The United States ◽

Test Results ◽

Pcr Assay ◽

Content Type ◽

Agar Dilution ◽

Ciprofloxacin Resistance ◽

To Receive

In the United States, 19.2% ofNeisseria gonorrhoeaeisolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive forN. gonorrhoeaeby the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

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Real-Time PCR Assay forClostridium perfringensin Broiler Chickens in a Challenge Model of Necrotic Enteritis

Applied and Environmental Microbiology ◽

10.1128/aem.01803-10 ◽

2010 ◽

Vol 77 (3) ◽

pp. 1135-1139 ◽

Cited By ~ 18

Author(s):

Shu-Biao Wu ◽

Nicholas Rodgers ◽

Mingan Choct

Keyword(s):

Regression Analysis ◽

Real Time ◽

Real Time Pcr ◽

Broiler Chickens ◽

Linear Regression Analysis ◽

Culture Method ◽

Necrotic Enteritis ◽

Pcr Assay ◽

Relative Standard ◽

Highly Correlated

ABSTRACTWe compared ilealClostridium perfringensquantification results produced by real-time PCR and culture-based methods in broiler chickens in a challenge model of necrotic enteritis. Assessment of the relative standard deviations (RSDs) revealed that the real-time PCR assay generated a smaller standard deviation and thus was more precise than the culture-based method. Linear regression analysis indicated that the bacterial counts of these two methods were highly correlated (R2= 0.845). We suggest that real-time PCR could be a replacement of the culture method for quantifyingC. perfringensin the intestinal tracts of broiler chickens.

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Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

Clinical Chemistry ◽

10.1093/clinchem/46.3.324 ◽

2000 ◽

Vol 46 (3) ◽

pp. 324-331 ◽

Cited By ~ 106

Author(s):

Danbing Ke ◽

Christian Ménard ◽

François J Picard ◽

Maurice Boissinot ◽

Marc Ouellette ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Culture Method ◽

Conventional Pcr ◽

Pcr Assay ◽

Group B Streptococci ◽

Standard Culture ◽

Pcr Assays ◽

Group B

Abstract Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

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Development of a Quantitative Real-Time PCR Method To Enumerate Total Bacterial Counts in Ready-to-Eat Fruits and Vegetables

Journal of Food Protection ◽

10.4315/0362-028x-69.10.2504 ◽

2006 ◽

Vol 69 (10) ◽

pp. 2504-2508 ◽

Cited By ~ 12

Author(s):

HAJIME TAKAHASHI ◽

HIROTAKA KONUMA ◽

YUKIKO HARA-KUDO

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fruits And Vegetables ◽

Bacterial Species ◽

Culture Method ◽

Plate Count ◽

Rrna Gene ◽

Pcr Assay ◽

Cycle Number ◽

Wide Range

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the β subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

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Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay

Journal of Medical Microbiology ◽

10.1099/jmm.0.45607-0 ◽

2004 ◽

Vol 53 (7) ◽

pp. 617-622 ◽

Cited By ~ 46

Author(s):

Yoshio Iijima ◽

Nahoko T. Asako ◽

Masanori Aihara ◽

Kozaburo Hayashi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bacterial Infections ◽

Food Poisoning ◽

Culture Method ◽

Pcr Analysis ◽

Laboratory System ◽

Pcr Assay ◽

Conventional Culture ◽

Stool Specimens

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40 %) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70 %) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18 %) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6 %) patients were found to be positive by PCR analysis.

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Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

Applied and Environmental Microbiology ◽

10.1128/aem.00517-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 6981-6985 ◽

Cited By ~ 26

Author(s):

Jianwei Huang ◽

Yumei Zhu ◽

Huixin Wen ◽

Jiafeng Zhang ◽

Shijie Huang ◽

...

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Toxin Gene ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Detection And Identification ◽

Life Threatening ◽

Toxigenic Strains

ABSTRACT Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.

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