scholarly journals Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools

2007 ◽  
Vol 56 (10) ◽  
pp. 1350-1355 ◽  
Author(s):  
Aisha Al Amri ◽  
Abiola C. Senok ◽  
Abdulrahman Yusuf Ismaeel ◽  
Ali E. Al-Mahmeed ◽  
Giuseppe A. Botta
Keyword(s):  
Multiplex Pcr ◽  
Direct Detection ◽  
False Negative ◽  
Enteric Bacteria ◽  
Enzymic Activity ◽  
Campylobacter Coli ◽  
Biochemical Tests ◽  
Direct Evaluation ◽  
Stool Specimens ◽  
Culture Negative

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015–0.016 ng C. jejuni and C. coli DNA μl−1 in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

scholarly journals Identification through Culture and Molecular Methods of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus in Surface Waters in Rasht

2019 ◽  
pp. 1-7
Author(s):  
Keyvan Roshanjo ◽  
Nematallah Jonaidi Jafari ◽  
Leila Asadpour ◽  
Reza Ranjbar ◽  
Davoud Afshar ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Campylobacter Jejuni ◽  
Surface Waters ◽  
Disease Transmission ◽  
Cross Sectional Study ◽  
Campylobacter Coli ◽  
Infectious Agents ◽  
Biochemical Tests ◽  
Cross Sectional ◽  
Campylobacter Fetus

Backgrounds: As zoonotic infectious agents, Campylobacter spp. are important factors causing gastroenteritis in humans. Surveys show that the three strains; Campylobacter jejuni, Campylobacter coli and Campylobacter fetus play a major role in human infections. Identification of these infectious agents is valuable for sanitary control of disease transmission through water resources. Objectives: The aim of this study was identification and molecular diagnosis of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus in surface waters in Rasht. Materials and Methods: This cross-sectional study was conducted on 45 samples of surface water in Rasht collected according to water health guidelines. After culture and biochemical tests on collected samples, detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus was done using sequence-specific amplification by Multiplex PCR. The results were subjected to statistical analysis using SPSS software. Results: Out of 45 samples tested, 6 were positive in culture, four of which were identified as Campylobacter jejuni after biochemical tests. Using Multiplex PCR, 8 samples were positive, from which 3 were Campylobacter jejuni, 1 Campylobacter coli and 4 were positive for both Campylobacter jejuni and Campylobacter coli. All the samples did not yield C. fetus. Conclusions: Multiplex PCR is regarded a diagnostic method with higher sensitivity and specificity than compared to methods for Campylobacter. The prevalence of Campylobacter jejuni and Campylobacter coli in surface waters in Rasht is considerable. Therefore, public health measures for the control of these organisms are recommended.

scholarly journals Multiplex PCR Assay for Identifi cation and Differentiation of Campylobacter jejuni and Campylobacter coli Isolates

Folia Medica ◽  
2016 ◽  
Vol 58 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Maria R. Pavlova ◽  
Elina G. Dobreva ◽  
Katucha I. Ivanova ◽  
Galina D. Asseva ◽  
Ivan N. Ivanov ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Campylobacter Jejuni ◽  
Accurate Method ◽  
Clinical Isolates ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Multiplex Pcr Assay ◽  
Hydrolysis Of ◽  
Campylobacter Spp

AbstractIntroduction: Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods.Aim: To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay.Materials and methods: Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli.Results and discussion: The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests.Conclusion: The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

scholarly journals Detection of 13 Enteric Bacteria and 5 Viruses Causing Acute Infectious Diarrhea Using Multiplex PCR from Direct Stool Specimens

2013 ◽  
Vol 16 (1) ◽  
pp. 33 ◽  
Author(s):  
Seungok Lee ◽  
Yeon-Joon Park ◽  
Hae Kyung Lee ◽  
Soo-Young Kim ◽  
Ja-Young Kim ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Enteric Bacteria ◽  
Infectious Diarrhea ◽  
Stool Specimens

scholarly journals PCR Detection of Escherichia coli O157:H7 Directly from Stools: Evaluation of Commercial Extraction Methods for Purifying Fecal DNA

2000 ◽  
Vol 38 (11) ◽  
pp. 4108-4113 ◽  
Author(s):  
J. L. Holland ◽  
L. Louie ◽  
A. E. Simor ◽  
M. Louie
Keyword(s):  
Escherichia Coli ◽  
False Negative ◽  
Extraction Methods ◽  
Rapid Identification ◽  
E Coli ◽  
Stool Specimens ◽  
Commercial Kits ◽  
Dna Template ◽  
Culture Positive ◽  
Culture Negative

Rapid identification of Escherichia coli O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract E. coli O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the E. coli O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.

scholarly journals Detection of LytA Genes in Streptococcus pneumoniae Isolated from sputum pneumonia patients

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23-30
Author(s):  
Mustika Sari Hutabarat ◽  
Firdaus Hamid ◽  
Irawaty Djaharuddin ◽  
Alfian Zainuddin ◽  
Rossana Agus ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
False Negative ◽  
Pneumococcal Infection ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Streptococcus pneumoniae (pneumococcus) is a Gram-positive facultative anaerobic bacterium that is a major cause of morbidity and mortality worldwide. But the lack of reporting of disease by this bacterium in Indonesia, one of the causes is because the diagnosis of pneumococcal infection is often clinically not typical and conventional methods which are still the standard gold method often give false-negative results. So the purpose of this study was to evaluate the performance of culture and molecular diagnostic methods using the Polymerase Chain Reaction (PCR) technique in detecting Streptococcus pneumoniae in sputum clinical samples using the Autolysin (LytA) gene which is a virulence factor of this bacterium. 57 isolates from 60 samples were confirmed as Streptococcus sp through microscopic identification, culture, and biochemical tests. Then the sensitivity test with an optochin test of 9 (9%) compared the results descriptively with the PCR technique using the Autolysin A (LytA) gene which was obtained more sensitive by 15 (25%).

scholarly journals Evaluation of a Multiplex PCR Assay for the Identification of Campylobacter jejuni and Campylobacter coli

2017 ◽  
Vol 3 (1) ◽  
pp. 6-8
Author(s):  
Saeed Shams ◽  
Mehdi Ghorbanalizadgan ◽  
Somayeh Haj Mahmmodi ◽  
Alessandra Piccirillo ◽  
◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Campylobacter Jejuni ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

scholarly journals Rapid Detection of Campylobacter coli, C. jejuni, and Salmonella enterica on Poultry Carcasses by Using PCR-Enzyme-Linked Immunosorbent Assay

2003 ◽  
Vol 69 (6) ◽  
pp. 3492-3499 ◽  
Author(s):  
Yang Hong ◽  
Mark E. Berrang ◽  
Tongrui Liu ◽  
Charles L. Hofacre ◽  
Susan Sanchez ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Salmonella Enterica ◽  
Rapid Detection ◽  
Diarrheal Disease ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Campylobacter Coli ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Serological Tests

ABSTRACT Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 � 102 and 4 � 101 CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for Salmonella. With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.

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