Detection of porcine circoviruses in clinical specimens using multiplex PCR in Hubei, central China

In order to detect and simultaneously discriminate PCV1, PCV2 and PCV3, a multiplex PCR assay was developed and used to detect clinical samples in this study. Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 and PCV3. The tissue samples from eight pig farms were detected using the multiplex PCR assay. The results showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and Real-time PCR methods. It proved that this multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei Province, Central China.

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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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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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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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Examining the Possibility of Detecting Brucella Canis from Tissue Samples Using Bruce-Ladder Multiplex PCR Assay

Acta veterinaria ◽

10.1515/acve-2017-0046 ◽

2017 ◽

Vol 67 (4) ◽

pp. 551-561

Author(s):

Nataša Stević ◽

Dušan Mišić ◽

Danica Bogunović ◽

Kazimir Matović ◽

Miroslav Valčić ◽

...

Keyword(s):

Multiplex Pcr ◽

Reproductive Organs ◽

Pcr Assay ◽

Tissue Samples ◽

Multiplex Pcr Assay ◽

Brucella Canis ◽

First Choice ◽

Two Samples ◽

Agglutination Method ◽

Canine Brucellosis

AbstractThe goal of this study was to compare the results of serological and conventional bacteriological methods with the results obtained using multiplex PCR Bruce-ladder assay. Based on the obtained results, the usability of the assay was assessed in regard to rapid diagnosis of canine brucellosis directly from the samples of reproductive organs of infected dogs. Out of 225 blood samples, 33 (14.67%) had a positive agglutination reaction. In this study, out of the 225 assayed reproductive organs of dogs, B. canis was isolated from 3 samples (1.33%), while the PCR Bruce-ladder assay detected two positive samples (0.88%). Two dogs from which B. canis was isolated, an antibody titer of 1/200 was established in blood serums, and third dog from which B. canis was isolated was negative using the tube agglutination test. From a total of 225 assayed organ samples, a positive PCR reaction was obtained from two samples. The obtained results show that the tube agglutination method remains the first choice for the detection of dogs infected with B. canis. In addition, whenever possible, it is necessary to try isolation. It is desirable to attempt the detection of B. canis in tissues using PCR, but the results may not be treated as definitive and reliable.

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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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A Multiplex PCR Assay Combined with Capillary Electrophoresis for the Simultaneous Identification of Atlantic Cod, Pacific Cod, Blue Whiting, Haddock, and Alaska Pollock

Foods ◽

10.3390/foods10112631 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2631

Author(s):

Yu-Min Lee ◽

Shinyoung Lee ◽

Hae-Yeong Kim

Keyword(s):

Capillary Electrophoresis ◽

Multiplex Pcr ◽

Atlantic Cod ◽

Limit Of Detection ◽

Pacific Cod ◽

Pcr Assay ◽

Food Fraud ◽

Blue Whiting ◽

Multiplex Pcr Assay ◽

Mitochondrial Cytochrome B

With an increased consumption of seafood products, food fraud with fish resources has been continuously reported. In particular, codfish has been exploited worldwide as a processed product in fresh, frozen, smoked, canned, or ready-to-eat dish forms. However, it is challenging to identify processed fish products after processing because of their similar morphological characteristics. Substitution and mislabeling of codfish among different species are also happening deliberately or unintentionally. Thus, it is necessary to distinguish cod species to prevent fish adulteration and food fraud. In this study, we developed a multiplex PCR for simultaneously identifying five cod species within Gadidae using capillary electrophoresis. Then, their species-specific primer sets were designed by targeting the mitochondrial cytochrome b gene. Subsequently, the amplicon sizes obtained were 237 bp, 204 bp, 164 bp, 138 bp, and 98 bp for Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock, respectively. The specificity of each primer was further tested using 19 fish species, and no cross-reactivity was observed. The limit of detection of this multiplex PCR assay was 1 pg. The developed multiplex PCR assay can be applied to 40 commercial food products successfully. This detection method will be efficient for managing seafood authentication by simultaneously analyzing multiple cod species.

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