Direct detection ofCampylobacter jejuniin human stool samples by real-time PCR

2008 ◽  
Vol 54 (9) ◽  
pp. 742-747 ◽  
Author(s):  
Shiyong Lin ◽  
Xinying Wang ◽  
Haoxuan Zheng ◽  
Zhengguo Mao ◽  
Yong Sun ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Culture Method ◽  
Turnaround Time ◽  
Culture Methods ◽  
Time Saving ◽  
Whole Process ◽  
Pure Cultures ◽  
Stool Samples

Our purpose was to establish a quick and accurate real-time PCR (rtPCR) method to detect Campylobacter jejuni directly from human diarrheal stool as an alternative to traditional culture methods. To determine the consistency of rtPCR and culture method, 256 clinical diarrheal stool samples and 50 normal stool samples from healthy individuals were examined, and the whole process was double-blinded. Our data showed that the sensitivity of rtPCR in pure cultures and stool was 102CFU·mL–1and 103CFU·g–1, respectively. Of the 256 diarrheal samples, 10 specimens were successfully detected by both methods, whereas two specimens were PCR positive but culture negative. No positive results were found by these two methods in 50 normal specimens. Our data suggested that rtPCR was convenient in operation and time-saving (turnaround time 3.5–4 h), so it could be used for clinical diagnostic and epidemiological purposes.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

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.

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

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
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.

Rapid, Specific Detection of Salmonella Enteritidis in Pooled Eggs by Real-Time PCR

2004 ◽  
Vol 67 (5) ◽  
pp. 864-869 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT ◽  
P. S. HOLT
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Specific Detection ◽  
Culture Methods ◽  
Conventional Culture ◽  
Low Concentrations ◽  
Pcr Method ◽  
Group D

An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5′ nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye–labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non–group D Salmonella and other non- Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 102 to 109 CFU/ml in phosphate-buffered saline and 103 to 108 CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.

Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods

2017 ◽  
Vol 20 (3) ◽  
pp. 477-484 ◽  
Author(s):  
M.A. Stachelska
Keyword(s):  
Real Time ◽  
Yersinia Enterocolitica ◽  
Real Time Pcr ◽  
Culture Method ◽  
Molecular Techniques ◽  
Pork Meat ◽  
Culture Methods ◽  
The Real ◽  
Pcr Method ◽  
Classical Culture

AbstractThe aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5’ nuclease PCR protocol. The probe was labelled at the 5’ end with the fluorescent reporter dye (FAM) and at the 3’ end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

scholarly journals Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens

2013 ◽  
Vol 62 (8) ◽  
pp. 1160-1164 ◽  
Author(s):  
Meng-Rui Lee ◽  
Kuei-Pin Chung ◽  
Hao-Chien Wang ◽  
Chih-Bin Lin ◽  
Chong-Jen Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Reference Method ◽  
Rapid Identification ◽  
Culture Methods ◽  
Cobas Taqman ◽  
Conventional Culture ◽  
Respiratory Specimens

The Cobas TaqMan MTB assay is a real-time PCR (qPCR) kit for rapid detection of Mycobacterium tuberculosis from clinical specimens. There are, however, limited studies validating its performance. We performed a prospective study in two hospitals in Taiwan on 586 respiratory specimens. By using culture as the reference method, the sensitivity and specificity of the Cobas TaqMan MTB assay were found to be 82.7 and 96.5 %, respectively. The sensitivity of the Cobas TaqMan MTB assay in acid-fast stain-negative respiratory specimens was only 34.9 %. Five specimens from five patients were positive for M. tuberculosis by the Cobas TaqMan MTB assay but were negative for M. tuberculosis by conventional culture methods. A diagnosis of pulmonary tuberculosis (TB) was made based on clinical and radiological findings as well as the response to anti-TB treatment in these five patients. Addition of data from these five specimens with discrepant results (PCR vs culture) from patients with symptoms clinically compatible with TB increased the sensitivity of the Cobas TaqMan MTB assay to 83.1 %. The Cobas TaqMan MTB assay is a rapid identification tool with a high degree of specificity for the direct detection of M. tuberculosis in respiratory specimens. The sensitivity for detecting acid-fast smear-negative respiratory specimens, however, is low.

scholarly journals Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed

2017 ◽  
Vol 80 (11) ◽  
pp. 1815-1820 ◽  
Author(s):  
Faiza Benahmed ◽  
Hua Wang ◽  
Junia Jean-Gilles Beaubrun ◽  
Gopal R. Gopinath ◽  
Chorng-Ming Cheng ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Culture Method ◽  
Whole Genome ◽  
Molecular Serotyping ◽  
Peptone Water ◽  
Pure Cultures

ABSTRACTBecause some significant outbreaks of human salmonellosis have been traced to contaminated animal feed, the rapid and efficient detection of Salmonella in feed is essential. However, the current U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) culture method that uses lactose broth as a preenrichment medium has not reliably supported the results of real-time PCR assays for certain foods. We evaluated the BAM culture method and a quantitative real-time PCR (qPCR) assay using two preenrichment media, modified buffered peptone water and lactose broth, to detect Salmonella enterica subsp. enterica serovar Cubana in naturally contaminated chick feed. After 24 h of incubation, the qPCR method was as sensitive as the culture method when modified buffered peptone water was used as the preenrichment medium but less sensitive than culture when lactose broth was used. After 48 h of incubation, detection of Salmonella Cubana by qPCR and by culture in either preenrichment medium was equivalent. We also compared the performance of the traditional serotyping method, which uses pure cultures of Salmonella grown on blood agar, to two molecular serotyping methods. The serotyping method based on whole genome sequencing also requires pure cultures, but the PCR-based molecular serotyping method can be done directly with the enriched culture medium. The PCR-based molecular serotyping method provided simple and rapid detection and identification of Salmonella Cubana. However, whole genome sequencing allows accurate identification of many Salmonella serotypes and highlights variations in the genomes, even in tight genomic clusters. We also compared the genome of the chick feed isolate with 58 Salmonella Cubana strains in GenBank and found that the chick feed isolate was very closely related to an isolate from a foodborne outbreak involving alfalfa sprouts.

A real-time PCR regimen for testing environmental samples for Salmonella enterica subsp. enterica serovars of concern to the poultry industry, with special focus on Salmonella Enteritidis

2019 ◽  
Vol 65 (2) ◽  
pp. 162-173 ◽  
Author(s):  
S. Nadin-Davis ◽  
L. Pope ◽  
D. Ogunremi ◽  
B. Brooks ◽  
J. Devenish
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Culture Collection ◽  
Special Focus ◽  
Culture Methods ◽  
Poultry Facilities ◽  
Culture Process

A real-time PCR (qPCR) regimen, using up to six genetic targets, was developed to rapidly detect Salmonella and in particular identify Salmonella Enteritidis. The test regimen was first evaluated using a reference culture collection of Salmonella to confirm the appropriateness of the selected targets, which included up to three genetic markers for discrimination of Salmonella Enteritidis from other Salmonella serovars commonly found in poultry facilities. The qPCR procedure was then compared with culture methods used to detect Salmonella using a collection of enrichment broths previously generated from 239 environmental samples collected from a large number of hatchery facilities across Canada over several years. The qPCR regimen facilitated specific detection of Salmonella Enteritidis, and on a sample basis, it showed excellent agreement with the culture methods. Moreover, in many cases, qPCR detected Salmonella earlier in the culture process than did the culture method. Application of this method will significantly shorten test times and allow more timely identification of infected poultry premises, thereby improving present programmes aimed at controlling Salmonella Enteritidis at the environmental source.

scholarly journals The Investigation of Streptococcus Agalactiae Colonization in Last Trimester Pregnants by Using Standard Culture and Molecular Methods

2021 ◽  
Author(s):  
Nurhadiye Kuru ◽  
Oguzhan Kuru ◽  
Abdullah Tüten ◽  
Nevriye Gonullu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Group B Streptococcus ◽  
Age Groups ◽  
Culture Method ◽  
Antibiotic Use ◽  
Vaginal Swab ◽  
Culture Methods ◽  
Cerrahpasa Medical Faculty ◽  
Significant Difference

Objective: We aimed to detect and compare group B streptococcus (GBS) colonization in pregnant women at third trimester using real-time polymerase chain reaction (real-time PCR) and culture methods. Methods: Vaginal swab specimens were taken for screening of GBS from 100 women between 35-37 weeks of gestation who were attending to antenatal outpatient unit of Obstetrics and Gynecology Department of Cerrahpasa Medical Faculty from May 2014 to September 2014. Results: Rates of GBS colonization was %5 and %7 by culture and real-time PCR methods, respectively. Using culture as the gold standard; sensitivity and specificity for real-time PCR were 100% and 97.9%, respectively. Any significant difference was not detected between GBS colonization with age groups, education levels, number of previous pregnancies, smoking habits, history of antibiotic use, and contraceptive method. Conclusion: Real-time PCR technique has proven to be as sensitive as the culture method. Also, real-time PCR may provide a rapid diagnostic tool for GBS detection potentially allowing a more effective intrapartum antibiotic prophylaxis and lower infant morbidity and mortality. However, the inability to use PCR test in every laboratory and its high cost creates a handicap.

scholarly journals Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

2000 ◽  
Vol 66 (10) ◽  
pp. 4266-4271 ◽  
Author(s):  
Hege Karin Nogva ◽  
Knut Rudi ◽  
Kristine Naterstad ◽  
Askild Holck ◽  
Dag Lillehaug
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Skim Milk ◽  
Raw Milk ◽  
Magnetic Bead ◽  
Quantitative Detection ◽  
Listeriolysin O ◽  
Pure Cultures

ABSTRACT PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenesbased on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all otherListeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

Salmonella detection in poultry samples

2012 ◽  
Vol 40 (06) ◽  
pp. 383-389 ◽  
Author(s):  
D. Sommer ◽  
D. Enderlein ◽  
A. Antakli ◽  
H. Schönenbrücher ◽  
J. Slaghuis ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Detection System ◽  
Cell Damage ◽  
Culture Method ◽  
Reference Laboratory ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Fecal Samples

Summary Study: The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 – Annex D) for the detection of Salmonella spp. in poultry samples. Material and methods: Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Results: Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100 cfu/25 g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14 cfu/25 g. Both real-time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. Clinical relevance: In general the advantage of PCR analyses over the culture method is the reduction of working time from 4–5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO 6579:2002 – Annex D.

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