Dataset Information

Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens.

ABSTRACT: Porcine respiratory disease complex (PRDC) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. No simple, single-reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Furthermore, the detection of most of the bacterial pathogens implicated in PRDC currently requires time-consuming culture-based methods that can take several days to obtain results. In this study, a novel prototype automated microarray that integrates and automates all steps of post-PCR microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with PRDC is described along with associated multiplex reverse transcriptase PCR. The user-friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus, porcine circovirus type 2, porcine respiratory corona virus], four bacteria (Mycoplasma hyopneumoniae, Pasteurella multocida, Salmonella enterica serovar Choleraesuis, Streptococcus suis), and further differentiated between type 1 and type 2 PRRSV as well as toxigenic and non-toxigenic P. multocida. The assay accurately identified and typed a panel of 34 strains representing the eight targeted pathogens and was negative when tested with 34 relevant and/or closely related non-target bacterial and viral species. All targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material.

SUBMITTER: Lung O

PROVIDER: S-EPMC7169873 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens.

Lung O O Ohene-Adjei S S Buchanan C C Joseph T T King R R Erickson A A Detmer S S Ambagala A A

Transboundary and emerging diseases 20151212 3

Porcine respiratory disease complex (PRDC) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. No simple, single-reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Furthermore, the detection of most of the bacterial pathogens implicated in PRDC currently requires time-consuming culture-based methods that can take several days to obtain results. In this study, ...[more]

PMID: 26662640

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Project description:A large number of viral and bacterial organisms are responsible for community-acquired pneumonia (CAP) which contributes to substantial burden on health management. A new resequencing microarray (RPM-IVDC1) associated with targeted multiplex PCR was recently developed and validated for multiple respiratory viruses detection and discrimination. In this study, we evaluated the capability of RPM-IVDC1 for simultaneous identification of multiple viral and bacterial organisms. The nasopharyngeal aspirates (NPAs) of 110 consecutive CAP patients, aged from 1 month to 96 years old, were collected from five distinct general hospitals in Beijing during 1-year period. The samples were subjected to the RPM-IVDC1 established protocol as compared to a real-time PCR (qRT-PCR), which was used as standard. The results of virus detection were consistent with those previously described. A total of 37 of Streptococcus pneumoniae, 14 of Haemophilus influenzae, 10 of Mycoplasma pneumoniae, two of Klebsiella pneumoniae and one of Moraxella catarrhalis were detected by RPM-IVDC1. The sensitivities and specificities were compared with those of qRT-PCR for S. pneumoniae (100, 100%, respectively), H. influenzae (92.3, 97.9%, respectively), M. pneumoniae (69.2, 99.0%, respectively), K. pneumoniae (100, 100%, respectively), and M. catarrhalis (100, 100%, respectively). Additional 22 of Streptococcus spp., 24 of Haemophilus spp. and 16 of Neisseria spp. were identified. In addition, methicillin-resistant and carbapenemases allele were also found in nine of Staphylococcus spp. and one of K. pneumoniae, respectively. These results demonstrated the capability of RPM-IVDC1 for simultaneous detection of broad-spectrum respiratory pathogens in complex backgrounds and the advantage of accessing to the actual sequences, showing great potential use of epidemic outbreak investigation. The detection results should be carefully interpreted when introducing this technique in the clinical diagnostics.

Project description:Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

Dataset Information

Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens.

Publications

Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens.

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