Development of multiplex PCR assay for rapid detection Listeria monocytogenes in clinical samples

Listeria monocytogenes is widely present in the natural environment. This bacteria can cause infections in both humans and animals. In humans, the most vulnerable groups to be infected with L. monocytogenes are the elderly, people with an impaired immune system and chronically illness, pregnant women, and newborn babies. The aim of this study was to develop a multiplex PCR assay for the rapid detection of L. monocytogenes in mock clinical samples. A pair of primers were designed for detection of L. monocytogenes based on prs, a Listeria genus specific gene, and hly, a hemolysin gene. The specificity of the primers were tested by using different L. monocytogenes strains and other common pathogenic bacteria. The results showed that L. monocytogenes strains were positive in the detection and other tested strains were negative in mock (spiked) clinical samples. The sensitivity of multiplex PCR assay was 102 CFU/ml per reaction. The specificity and sensitivity of multiplex PCR technology for detecting L. monocytogenes in mock (spiked) clinical samples were high, and the assay could be completed within 1.5 hours. Therefore, this established multiplex PCR provides a rapid and reliable method and will be useful for the detection of L. monocytogenes in mock clinical samples.

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Rapid detection of Listeria monocytogenes sequence type 121 strains using a novel multiplex PCR assay

LWT ◽

10.1016/j.lwt.2019.108474 ◽

2019 ◽

Vol 116 ◽

pp. 108474 ◽

Cited By ~ 2

Author(s):

Moutong Chen ◽

Jianheng Cheng ◽

Rui Pang ◽

Jumei Zhang ◽

Yuetao Chen ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Rapid Detection ◽

Sequence Type ◽

Pcr Assay ◽

Multiplex Pcr Assay

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Development of multiplex PCR for rapid detection of metallo-β-lactamase genes in clinical isolates of Acinetobacter baumannii

Iranian Journal of Microbiology ◽

10.18502/ijm.v12i2.2615 ◽

2020 ◽

Author(s):

Reza Ranjbar ◽

Shahin Zayeri ◽

Amir Mirzaie

Keyword(s):

Acinetobacter Baumannii ◽

Multiplex Pcr ◽

Rapid Detection ◽

Simultaneous Detection ◽

Cross Reactivity ◽

Clinical Isolates ◽

Clinical Samples ◽

Pcr Assay ◽

Multiplex Pcr Assay

Background and Objectives: Acinetobacter baumannii has been known as a major pathogen causing nosocomial infec- tions. The aim of this study was to develop multiplex PCR for rapid and simultaneous detection of metallo-β-lactamase (MBL) genes in clinical isolates of A. baumannii. Materials and Methods: In this study, we used three sets of primers to amplify the MBL genes including bla , bla and bla OXA-48 . The multiplex PCR assay was optimized for rapid and simultaneous detection of MBL genes in A. bau- OXA-23 NDM mannii strains recovered from clinical samples. Results: A. baumannii strains recovered from clinical samples were subjected to the study. The multiplex PCR produced 3 OXA-48 OXA-23 bands of 501 bp for bla , 744 bp for bla observed in multiplex PCR. OXA-48 and 623 bp for bla NDM genes. In addition to, no any cross-reactivity was Conclusion: Based on obtained data, the multiplex PCR had a good specificity without any cross reactivity and it appears that the multiplex PCR is reliable assay for simultaneous detection of MBL genes in A. baumannii strains.

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Evaluation of a Serotyping Scheme Using a Combination of an Antibody-Based Serogrouping Method and a Multiplex PCR Assay for Identifying the Major Serotypes of Listeria monocytogenes

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-355 ◽

2011 ◽

Vol 74 (3) ◽

pp. 403-409 ◽

Cited By ~ 28

Author(s):

LAUREL S. BURALL ◽

ALEXANDRA C. SIMPSON ◽

ATIN R. DATTA

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Gold Standard ◽

Combination Method ◽

Clinical Samples ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Combination Scheme ◽

Hlya Gene

To evaluate a simplified serotyping scheme, we used a combination of an antibody-based serogrouping assay that identified only type 1 and type 4 strains and a multiplex PCR–based serogrouping assay to analyze 362 L. monocytogenes isolates collected over more than 20 years. The multiplex PCR assay also incorporated a set of primers specific for L. monocytogenes hlyA gene to verify the species identification of these isolates. A subset (n = 120) of these isolates were also serotyped with the Denka Seiken serotyping scheme, which is often considered the “gold standard” for serotyping of L. monocytogenes. The results indicate that the multiplex PCR–based assay, in combination with an antibody-based serogrouping assay, correctly identified serotypes of 96% of the previously serotyped isolates. Compared with the Denka Seiken method, the combination method also performed better in identifying serotypes of 120 previously unserotyped L. monocytogenes isolates. Thus, the combination scheme appears to be a simple and rapid way to identify serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 4b isolates, which are the predominant L. monocytogenes serotypes found in food, environmental, and clinical samples.

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Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry

AMB Express ◽

10.1186/s13568-019-0908-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Zhihao Wang ◽

Jiakun Zuo ◽

Jiansen Gong ◽

Jiangang Hu ◽

Wei Jiang ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Pasteurella Multocida ◽

Target Genes ◽

Bacterial Pathogens ◽

Clinical Samples ◽

Test Results ◽

Pcr Assay ◽

Specific Test ◽

Multiplex Pcr Assay ◽

Specificity And Sensitivity

AbstractEscherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8–8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

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Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay

Journal of Virological Methods ◽

10.1016/j.jviromet.2008.11.007 ◽

2009 ◽

Vol 156 (1-2) ◽

pp. 111-116 ◽

Cited By ~ 82

Author(s):

Suk Ran Kim ◽

Chang-Seok Ki ◽

Nam Yong Lee

Keyword(s):

Multiplex Pcr ◽

Rapid Detection ◽

Respiratory Viruses ◽

Pcr Assay ◽

Dual Priming Oligonucleotide ◽

Multiplex Pcr Assay ◽

Detection And Identification

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Multiplex PCR Assay for Rapid Detection and Genotyping of Helicobacter pylori Directly from Biopsy Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.42.6.2821-2824.2004 ◽

2004 ◽

Vol 42 (6) ◽

pp. 2821-2824 ◽

Cited By ~ 56

Author(s):

S. Chattopadhyay ◽

R. Patra ◽

T. Ramamurthy ◽

A. Chowdhury ◽

A. Santra ◽

...

Keyword(s):

Helicobacter Pylori ◽

Multiplex Pcr ◽

Rapid Detection ◽

Pcr Assay ◽

Biopsy Specimens ◽

Multiplex Pcr Assay

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Evaluation of the AllplexTM Gastrointestinal Panel—Parasite Assay for Protozoa Detection in Stool Samples: A Retrospective and Prospective Study

Microorganisms ◽

10.3390/microorganisms8040569 ◽

2020 ◽

Vol 8 (4) ◽

pp. 569 ◽

Cited By ~ 1

Author(s):

Brice Autier ◽

Jean-Pierre Gangneux ◽

Florence Robert-Gangneux

Keyword(s):

Prospective Study ◽

Multiplex Pcr ◽

Clinical Samples ◽

Molecular Assay ◽

Pcr Assay ◽

Cryptosporidium Spp ◽

Multiplex Pcr Assay ◽

Parasite Detection ◽

Stool Samples ◽

Higher Sensitivity

This study aims at evaluating the performances of the multiplex PCR AllplexTM Gastrointestinal Panel-Parasite Assay (GIPPA), which detects G. duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, B. hominis, and C. cayetanensis, by comparison to microscopy. A retrospective evaluation was conducted on a series of positive clinical samples (n = 99) stored at −80 °C or at +4 °C. A five-month prospective study was then conducted on all samples sent to our lab for parasite detection (n = 586). In the retrospective cohort, sensitivity was 81% for both G. duodenalis (26/32) and D. fragilis (21/26) and 100% for Cryptosporidium spp. (26/26, including 6 different species), B. hominis (26/26), and C. cayetanensis (4/4). During the prospective study, 95 samples were positive by microscopy and 207 by multiplex PCR assay. The molecular assay showed a significantly higher sensitivity of PCR, especially for G. duodenalis (100% vs. 60.7%, p < 0.01), D. fragilis (97.2% vs. 14.1%, p < 0.001), and B. hominis (99.4% vs. 44.2%, p < 0.001) but also for E. histolytica (100% vs. 50.0%). The sensitivity of the AllplexTM GIPPA on the first stool sample was equivalent to the sensitivity of microscopy on multiple stool samples but inferior to multiplex PCR on multiple stool samples. Taken together, the AllplexTM GIPPA is suitable for the routine detection of protozoa in fecal samples.

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

Journal of Food Protection ◽

10.4315/0362-028x-65.5.780 ◽

2002 ◽

Vol 65 (5) ◽

pp. 780-785 ◽

Cited By ~ 30

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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