Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid

Wisselink, Henk J.; Cornelissen, J.B.W.J.; Wal, F.J. van der; Kooi, Engbert A.; Koene, Miriam; Bossers, Alex; Smid, Bregtje; Bree, F.M. de; Antonis, A.F.G.

doi:10.1186/s12917-017-1141-1

Cited by 14 publications

(8 citation statements)

References 26 publications

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“…Pneumo4B and Pneumo4V were able to detect between 10 copies of genomic DNA and 10-50 copies of target RNA per reaction, respectively, which are comparable to previously established methods which reported of 100-250 copies per reaction (Rahpaya et al, 2018). The qPCR efficiency was also similar to multiplex qPCR assays reported by others Wisselink et al, 2017).…”

Section: Discussionsupporting

confidence: 84%

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

Pansri

Katholm

Krogh

et al. 2020

The Veterinary Journal

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A B S T R A C TBovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively.Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93-106% and 91-104%, respectively, with assay detection limits of 10-50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135 strains of non-target bacterial species. Based on standard curves of log 10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71-0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

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Section: Discussionsupporting

confidence: 84%

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

Pansri

Katholm

Krogh

et al. 2020

The Veterinary Journal

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“…However the authors also stated that the best method to detect and identify M. bovis was also dependent on the sample type. Here, the naturally infected BALFs contained several other bacteria [ 34 ] including mycoplasmas [ 22 ] among which M. bovirhinis , which is a known fast grower. Hence, M. bovirhinis present in the sample could overgrow M. bovis and lead to false negative results depending on the techniques used afterwards to differentiate M. bovis from M. bovirhinis .…”

Section: Discussionmentioning

confidence: 99%

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

et al. 2019

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BackgroundSeveral species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis.ResultsThe participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories.ConclusionWith a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.Electronic supplementary materialThe online version of this article (10.1186/s12917-019-1819-7) contains supplementary material, which is available to authorized users.

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“…The PCR method has been widely used in laboratories and hospitals. In order to detect the target gene of pathogens, it needs to extract and purify the genome in the sample first, and then detect the target gene with specific primers (Wisselink et al 2017). The operation is complicated, and it takes 1-2 h to obtain the result.…”

Section: Diagnostic Methods For Public Health Emergenciesmentioning

confidence: 99%

Application of UPT-POCT in Public Health Emergencies

Li¹,

Ren²,

Jia³

et al. 2019

Principles and Applications of Up-Converting Phosphor Technology

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Rapid and reliable detection of infectious agents on site is essential for timely initiation of medical treatment and post-exposure prophylactic measures when public health emergencies occur. However, the referee standard for confirmation of infectious agents remains laboratory diagnosis, which is time-consuming and not available in the field. UPT-POCT technology is a versatile tool that requires limited resources and can realize rapid detection of infectious agents on site, providing timely information for the quick response to public health emergencies.

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Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid

Cited by 14 publications

References 26 publications

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

Application of UPT-POCT in Public Health Emergencies

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