Comparison of Culture, Multiplex, and 5′ Nuclease Polymerase Chain Reaction Assays for the Rapid Detection of Yersinia enterocolitica in Swine and Pork Products†

2001 ◽  
Vol 64 (9) ◽  
pp. 1352-1361 ◽  
Author(s):  
SANDHYA BOYAPALLE ◽  
IRENE V. WESLEY ◽  
H. SCOTT HURD ◽  
P. GOPAL REDDY
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Culture Method ◽  
Taqman Assay ◽  
Chain Reaction ◽  
Pork Products ◽  
Ground Pork ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Pork Samples

Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected 1 ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 × 103, 4 × 102, and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 × 102, 4 × 102, and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

Diagnostic Value of Multiplex Polymerase Chain Reaction in Detection of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from Sepsis in Pediatrics

2021 ◽  
Vol 15 ◽  
Author(s):  
Sara Galeb ◽  
Maysaa El Sayed Zaki ◽  
Raghdaa Shrief ◽  
Rasha Hassan ◽  
Mohamed Anies
Keyword(s):  
Polymerase Chain Reaction ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Multiplex Pcr ◽  
Stenotrophomonas Maltophilia ◽  
Multiplex Polymerase Chain Reaction ◽  
Diagnostic Value ◽  
Blood Cultures ◽  
Chain Reaction ◽  
Polymerase Chain

Background: Proper identification of the causative organism in pediatric sepsis is crucial for early diagnosis and prevention of septic shock and organ failure. Objectives: The present study aimed to evaluate the multiplex Polymerase Chain Reaction (PCR) to detect Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures for these pathogens isolated from children, with hospital-acquired sepsis compared to the conventional biochemical reactions for identification of these organisms. Methods: This study was a cross-sectional study performed on 100 isolates from pediatric blood cultures, including Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The study also included 100 isolates of Escherichia coli as a negative control. All isolates were identified by API 20NE and the multiplex PCR, with primers specific to the 3 tested bacteria. Results: Multiplex PCR was positive in 96% of isolates, and 4 isolates had negative results. False positive results were reported with three E. coli strains. Multiplex PCR identified all the isolates of Acinetobacter baumannii, 29 isolates of Pseudomonas aeruginosa, and 27 isolates of Stenotrophomonas maltophilia. Compared to the biochemical identification, the diagnostic value of the multiplex PCR revealed 96.04% sensitivity, 96.9% specificity, 97.00%, positive predictive value, 96.00% negative predictive value, and 96.50% accuracy. Conclusion: The present study highlights the diagnostic value of multiplex PCR to identify Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures. Multiplex PCR was sensitive, specific, and accurate. The accuracy differs according to the organisms, with 100% accuracy for Acinetobacter baumannii.

Development of a Multiplex Polymerase Chain Reaction Assay for Detection and Differentiation of Staphylococcus aureus in Dairy Products†

2001 ◽  
Vol 64 (5) ◽  
pp. 664-668 ◽  
Author(s):  
SUDHIR TAMARAPU ◽  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Dairy Products ◽  
Multiplex Polymerase Chain Reaction ◽  
Skim Milk ◽  
Cheddar Cheese ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

scholarly journals Prevalence and diversity of Arcobacter spp. in retail chicken meat in Turkey

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Celenk Molva ◽  
Halil Ibrahim Atabay
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Chicken Meat ◽  
Species Level ◽  
Waterborne Pathogens ◽  
Chain Reaction ◽  
Retail Markets ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Retail Chicken Meat

Arcobacters are food and waterborne pathogens associated with human and animal infections. The objective of the present study was to investigate the prevalence and diversity of <em>Arcobacter</em> spp. in commercially sold chicken meat in İzmir region of Turkey. For this purpose, 100 samples including legs (n=40), 17 chicken quarters (n=17), drumstickers (n=16), breasts (n=11), wings (n=10), and carcasses (n=6) were collected from different retail markets. A total of 65 isolates were confirmed as <em>Arcobacter</em> spp. from 55 samples by genus-specific polymerase chain reaction (PCR). The prevalence of <em>Arcobacter</em> spp. was 32.5, 81.3, 64.7, 72.7, 83.3, and 50% for legs, drumstickers, chicken quarters, breasts, carcasses and wings, respectively. Based on the multiplex-PCR, most of the isolates were identified as <em>A. butzleri</em> (n=45, 80%), followed by <em>A. cryaerophilus</em> (n=2, 3.6%), <em>A. skirrowii</em> (n=1, 1.8%) and 17 isolates (30.9%) could not be identified at the species level.

scholarly journals Molecular identification by multiplex polymerase chain reaction of Pasteurella multocida in cattle and buffaloes in Baghdad

2014 ◽  
Vol 38 (1) ◽  
pp. 99-106
Author(s):  
Ihab G. M. AL-Shemmari
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Pasteurella Multocida ◽  
Type B ◽  
Chain Reaction ◽  
Blood Samples ◽  
Nasal Swabs ◽  
Polymerase Chain ◽  
B 31

The aim of this study was to identify pasteurella multocida and their types by PCR in cattle’s and buffaloesi bagdad from March to August 2012 on 204 animals , including 102 cattle and 102 buffaloes at slaughter houses from Baghdad .Blood samples and nasal swaps were collected , before slaughtering and lung tissues of slaughtered animal , and from 54 clinically suspected cases of pasteurellosis , including 27 bovines ,and 27 buffaloes the samples taken included blood and nasal swabs . Pasteurellamultocida were isolated from 94 animals include 49 cattle 45 buffaloes. The typing of the isolates by multiplex PCR for genotyping Pasteuerllamultocida revealed 93 isolates of type B , 31 from cattle and 62 from buffaloes ,and 81 isolates of type A , 55 from cattle and 26 from buffaloes .

scholarly journals Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

2002 ◽  
Vol 65 (5) ◽  
pp. 780-785 ◽  
Author(s):  
IRENE V. WESLEY ◽  
KAREN M. HARMON ◽  
JAMES S. DICKSON ◽  
ANN RAMOS SCHWARTZ
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Listeria Species ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

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