Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 103 c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

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

Tạp chí Y học Dự phòng ◽

10.51403/0868-2836/2020/246 ◽

2021 ◽

Vol 30 (4) ◽

pp. 20-26

Author(s):

Le Thanh Huong ◽

Ha Thi Phuong Mai ◽

Hoang Thi Thu Ha ◽

Nguyen Dong Tu ◽

Bui Tien Sy ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Rapid Detection ◽

Pathogenic Bacteria ◽

Vulnerable Groups ◽

Clinical Samples ◽

Specific Gene ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Specificity And Sensitivity

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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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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Prevalence and Diversity of Arcobacter spp. in the Czech Republic

Journal of Food Protection ◽

10.4315/0362-028x-71.4.719 ◽

2008 ◽

Vol 71 (4) ◽

pp. 719-727 ◽

Cited By ~ 21

Author(s):

M. PEJCHALOVÁ ◽

E. DOSTALÍKOVÁ ◽

M. SLÁMOVÁ ◽

I. BROŽKOVÁ ◽

J. VYTŘASOVÁ

Keyword(s):

Surface Water ◽

Multiplex Pcr ◽

Chicken Meat ◽

Clinical Samples ◽

Rrna Gene ◽

Retail Market ◽

Meat Processing ◽

Chicken Carcass ◽

Fish Samples ◽

Chicken Breeds

The aim of this study was to examine 634 samples of chicken, lamb, pork, beef, fish, samples from the intensive animal industry and from poultry for slaughter, as well as from the domestic breeding of poultry, horses, pigs, and lambs, from surface water, and from clinical samples for the presence of Arcobacter. All the samples were examined with a cultivation method, followed by confirmation by multiplex PCR. The method of multiplex PCR applied directly to a liquid medium after enrichment was applied only to the samples with the highest probability of the presence of arcobacters. Arcobacter spp. were detected in 11.8% of the samples, of which A. butzleri, A. cryaerophilus, and A. skirrowii were found in 6.6, 5.1, and 0.2% of the samples, respectively. The sources of the arcobacters were chicken meat from the retail market, intensive animal production facilities, domestic chicken breeding facilities, lamb raising environments, surface water and wastewater, and beef swabs taken in a meat processing factory. No occurrence of arcobacters was identified in the swabs from slaughter turkeys, ducks, and wild poultry. No arcobacters were found in horse and pig breeding environments, on pork, or on the swabs of fish. Forty-two rectal swabs taken from humans were also free of Arcobacter. Seventeen isolates of Arcobacter were further identified by sequencing the 16S rRNA gene. Varied genotypes were observed among A. butzleri from chicken meat and chicken breeds, and A. cryaerophilus from wastewater and chicken breeds. They were similar to the genotypes present in wastewater, porcine feces, human stool, and human blood obtained from databases. Our results revealed that the chicken meat from the retail market is an important source of arcobacters. Cross-contamination during handling of chicken carcass practices could play a key role in the spread of Arcobacter.

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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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A Novel Multiplex-PCR Assay to Detect Three Non-Halal Meats Contained in Meatball using Mitochondrial 12S rRNA Gene

Food Science of Animal Resources ◽

10.5851/kosfa.2020.e40 ◽

2020 ◽

Vol 40 (4) ◽

pp. 628-635

Author(s):

Muhammad Cahyadi ◽

Tommy Wibowo ◽

Ahmad Pramono ◽

Zakaria Husein Abdurrahman

Keyword(s):

Multiplex Pcr ◽

12S Rrna ◽

Rrna Gene ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

12S Rrna Gene ◽

Mitochondrial 12S Rrna

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Detection of Nocardia, Streptomyces and Rhodococcus from bronchoalveolar lavage specimens of patients with HIV by Multiplex PCR Assay

Ethiopian Journal of Health Sciences ◽

10.4314/ejhs.v29i6.10 ◽

1970 ◽

Vol 29 (6) ◽

Author(s):

Hossein Ali Rahdar ◽

Mohammad Reza Salehi ◽

Abass Bahador ◽

Seyedesomaye Jasemi ◽

Morteza Karami-Zarandi ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Multiplex Pcr ◽

Target Genes ◽

Pcr Assay ◽

Accurate Identification ◽

Streptomyces Spp ◽

Multiplex Pcr Assay ◽

Pcr Products ◽

Pure Cultures ◽

Immune Health

Background: Nocardia, Streptomyces and Rhodococcus are life threatening opportunistic pathogens under immunodeficiency conditions, particularly among patients infected with HIV. Rapid and accurate detection of these infections can improve immune health quality, patient management and appropriate treatment. The aim of this study was to design a novel multiplex-PCR assay for rapid diagnosis of these three organisms directly from bronchoalveolar lavage (BAL) specimens of patients infected with HIV.Methods: The genus specific primers were designed for directdetection of Nocardia, Streptomyces and Rhodococcus in a single tube multiplex PCR. This PCR specifically amplified the target genes from pure cultures. It subsequently was applied on BAL specimens of 29 HIV positive patients that had previously been culture negative for actinomycete bacteria, of which Nocardia, Streptomyces and Rhodococcus are members.Results: Of 29 respiratory clinical specimens, there were positive for Nocardia spp. and one was positive for Streptomyces spp using the multiplex PCR assay. The sequencing of the PCR products identified the species as Nocardia cyriacigeorgica (n=2), Nocardia farcinica and Streptomyces albus.Conclusion: This novel multiplex PCR assay yielded reliable results for accurate identification of Nocardia, Streptomyces and Rhodococcus from BAL while the results of bacterial culture were negative.

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Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

Journal of Veterinary Diagnostic Investigation ◽

10.1177/10406387211063438 ◽

2021 ◽

pp. 104063872110634

Author(s):

Barbara Ujvári ◽

Hubert Gantelet ◽

Tibor Magyar

Keyword(s):

Multiplex Pcr ◽

Pasteurella Multocida ◽

Rrna Gene ◽

Pcr Assay ◽

Biochemical Tests ◽

Specific Primers ◽

Multiplex Pcr Assay ◽

Subspecies Level ◽

Species Specific ◽

Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

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Application of a multiplex PCR assay for the detection ofShigella,Escherichia coliand Shiga toxin-producingEsch. coliin milk

Journal of Dairy Research ◽

10.1017/s0022029909004026 ◽

2009 ◽

Vol 76 (2) ◽

pp. 188-194 ◽

Cited By ~ 14

Author(s):

Syed Riyaz-Ul-Hassan ◽

Saima Syed ◽

Sarojini Johri ◽

Vijeshwar Verma ◽

Ghulam Nabi Qazi

Keyword(s):

Multiplex Pcr ◽

Shiga Toxin ◽

Raw Milk ◽

Pcr Assay ◽

Milk Samples ◽

Multiplex Pcr Assay ◽

The Common ◽

Different Strains ◽

Mpcr Assay ◽

Hygienic Conditions

A multiplex PCR (mPCR) assay using previously known genetic markers ofShigella, Escherichia coliand Shiga-toxicEsch. coliwas standardized.uidAgene was targeted for the common detection ofEsch. coliandShigella, whereasipaHandstx1genes were used as markers for the detection ofShigellaand shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of theipaHanduidAgene fragments indicated the presence ofShigellaspp., amplification ofuidAalone revealed the presence ofEsch. coliand additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains ofEsch. coliandShigellaspp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-μl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100),Esch. coliwere detected in all samples and verotoxinogenicEsch. coliin 15 samples.Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxicEsch. coliwas directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies.

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