Development of a novel detection system for microbes from bovine diarrhea by real-time PCR

Tsuchiaka, Shinobu; Masuda, Tsuneyuki; Sugimura, Satoshi; Kobayashi, Suguru; Komatsu, Natsumi; Nagai, Makoto; Omatsu, Tsutomu; Furuya, Tetsuya; Oba, Mami; Katayama, Yukie; Kanda, Shuhei; Yokoyama, Tadashi; Mizutani, Tetsuya

doi:10.1292/jvms.15-0552

Cited by 28 publications

(53 citation statements)

References 29 publications

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“…Although several molecular assays have been developed to detect multiple BED pathogens (Fukuda et al, 2012;Asano et al, 2010;Loa et al, 2006;Tsuchiaka et al, 2016), to our knowledge, the viral and bacterial/protozoa assays described here are the most comprehensive assays published to date for detection and differentiation of BED causative pathogens in a single reaction. A multiplex PCR assay for detection of five BED-associated viruses, namely BCoV, BRoV (strains A, B, and C) and BToV has been described previously (Fukuda et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…Recently several multiplex real-time PCR assays have been developed to detect pathogens associated with BRDC and BED (Horwood and Mahony, 2011;Marley et al, 2008;Thonur et al, 2012;Cho et al, 2010). Alternatively, a real-time PCR based system was described that includes multiple reactions in one-run with similar thermal cycling conditions for detection of BRDC and BED pathogens (Kishimoto et al, 2017;Tsuchiaka et al, 2016). Although these real-time PCR assays are rapid and highly sensitive, their multiplexing capability at present is restricted by the availability of limited number of compatible fluorescent dyes.…”

Section: Introductionmentioning

confidence: 99%

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A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Thanthrige-Don

Lung

Furukawa-Stoffer

et al. 2018

Journal of Virological Methods

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Thanthrige-Don

Lung

Furukawa-Stoffer

et al. 2018

Journal of Virological Methods

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“…Most tests are based on a one assay-one pathogen approach, and they are not enough for diagnosis of PRDC in terms of comprehensiveness and rapidity. Tsuchiaka et al previously developed a system to detect microbes in bovine diarrhea by TaqMan real-time PCR, permitting the simultaneous screening of 19 pathogens associated with diarrhea [26]. TaqMan real-time PCR possesses the advantages of high sensitivity, high specificity, and simple operation.…”

mentioning

confidence: 99%

Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases

Sunaga

Tsuchiaka

Kishimoto

et al. 2020

The Journal of Veterinary Medical Science

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The etiology of Porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. In this present study, we developed a detection system of microbes from porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. We selected 17 porcine respiratory pathogens (Actinobacillus pleuropneumoniae, Boldetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Pasteurella multocida toxin, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovie, porcine circovirus 2, pseudorabies virus, porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. However, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory symptoms on six farms were tested by the Dempo-PCR to validate the assay's clinical performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and porcine reproductive and respiratory virus US strain, Mycoplasma hyorhinis, Haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. These results suggest that Dempo-PCR assay can be applied as a screening system with wide detection targets.

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“…To confirm the presence of other pathogens in the Kagoshima sample, detection of agents causing diarrhea using our real-time PCR system, referred to as “Dembo-PCR,” was performed [46]. This system can identify 19 species of pathogens, including virus, bacteria and protozoa.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, viral DNA and RNA were extracted by high pure viral nucleic acid extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) and bacteria and protozoa DNA were extracted by QIAamp Fast DNA stool mini kit (QIAGEN, Hilden, Germany). Nucleic acids extracted by each kit were subjected to Dembo-PCR, according to a previous report [46]. …”

Section: Methodsmentioning

confidence: 99%

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein

et al. 2017

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BackgroundBovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5′ untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted.ResultsThe complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5′UTR, open reading frame encoding a single polyprotein, and 3′UTR. The results of secondary RNA structure analysis showed that in the 5′UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region.ConclusionsWe identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as “Enterovirus K”, which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0923-0) contains supplementary material, which is available to authorized users.

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Development of a novel detection system for microbes from bovine diarrhea by real-time PCR

Cited by 28 publications

References 29 publications

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein

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