A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses

Shi, Xinjie; Liu, Xuming; Wang, Qin; Das, Asish R.; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei Hua; Liu, Yanhua; Anderson, Gary A.; Bai, Jianfa; Shi, Jishu

doi:10.1016/j.jviromet.2016.08.005

Cited by 31 publications

(45 citation statements)

References 32 publications

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“…Additionally, multiple tests are required to detect coinfections or to differentiate between species with clinically indistinguishable signs. Although multiplex real‐time RT‐PCRs have been described for many swine pathogens (Haines et al., ; Li et al., ; Rasmussen et al., ; Shi et al., ; Wernike et al., , ), the diagnosis of FMDV, SVDV, CSFV and ASFV still requires the performance of multiple single tests. Automated multiplex assays can potentially reduce labour cost and handling time.…”

Section: Discussionmentioning

confidence: 99%

“…User‐friendly reverse transcription insulated isothermal PCR (RT‐iiPCR) assays performed on compact, field‐deployable instruments with automatic display of “+” or “−” results have also been described for CSFV (Lung et al., ) and FMDV (Ambagala et al., ). Real‐time multiplex PCR assays have been reported for the detection of FMDV and CSFV (Shi et al., ; Wernike et al., ) and other targets (Haines, Hofmann, King, Drew, & Crooke, ; Li et al., ; Rasmussen et al., ; Wernike et al., ). Although RT‐iiPCR and real‐time RT‐PCR are rapid and highly sensitive, separate single‐target reactions are normally performed by the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease when testing for the presence of foreign animal disease pathogens.…”

Section: Introductionmentioning

confidence: 99%

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A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

Erickson

Fisher

Furukawa-Stoffer

et al. 2017

Transbound Emerg Dis

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Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

Erickson

Fisher

Furukawa-Stoffer

et al. 2017

Transbound Emerg Dis

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show abstract

“…Polymerase chain reaction tests for diagnosis were performed as described (Fowler et al, ; Shi et al, ). Briefly, a master mix of a 20 µl reaction is composed of 5 µl of template RNA, 0.4 µM each of species‐specific forward and reverse PCR primers, 0.2 µM each of probes, and 10 µl reaction buffer and 2 µl of enzyme mix from Path‐ID™ Multiplex One‐Step RT‐PCR Kit (ThermoFisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…The presence of cloned inserts was confirmed by gel electrophoresis and Sanger sequencing. The positive standards for the 3D‐based SVV‐1 assay and the FMDV assay in the Shi et al assay were either amplified (SVV‐1) or synthesized (FMDV) and cloned in the previous studies (Fowler et al, ; Shi et al, ). As both the Shi assay and the Callahan assay are using a similar region for primer designs, the positive amplification control works for both assays.…”

Section: Methodsmentioning

confidence: 99%

Development and evaluation of multiplex real‐time RT‐PCR assays for the detection and differentiation of foot‐and‐mouth disease virus and Seneca Valley virus 1

Wang

Das

Zheng

et al. 2019

Transbound Emerg Dis

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Foot‐and‐mouth disease virus (FMDV) causes a highly contagious and economically important vesicular disease in cloven‐hoofed animals that is clinically indistinguishable from symptoms caused by Seneca Valley virus 1 (SVV‐1). To differentiate SVV‐1 from FMDV infections, we developed a SVV‐1 real‐time RT‐PCR (RT‐qPCR) assay and multiplexed with published FMDV assays. Two published FMDV assays (Journal of the American Veterinary Medical Association, 220, 2002, 1636; Journal of Virological Methods, 236, 2016, 258) targeting the 3D polymerase (3D) region were selected and multiplexed with the SVV‐1 assay that has two targets, one in the 5′ untranslated region (5′ UTR, this study) and the other in the 3D region (Journal of Virological Methods, 239, 2017, 34). In silico analysis showed that the primers and probes of SVV‐1 assay matched 98.3% of the strain sequences (113/115). The primer and probe sequences of the Shi FMDV assay matched 85.4% (806/944), and that of the Callahan FMDV assay matched 62.7% (592/944) of the sequences. The limit of detection (LOD) for the two multiplex RT‐qPCR assays for SVV‐1 was both 9 copies per reaction by cloned positive plasmids and 0.16 TCID50 per reaction by cell culture. The LOD for FMDV by both multiplex assays was 11 copies per reaction using cloned positive plasmids. With cell cultures of the seven serotypes of FMDV, the Shi assay (Journal of Virological Methods, 236, 2016, 258) had LODs between 0.04 and 0.18 TCID50 per reaction that were either the same or lower than the Callahan assay. Interestingly, multiplexing with SVV‐1 increased the amplification efficiencies of the Callahan assay (Journal of the American Veterinary Medical Association, 220, 2002, 1636) from 51.5%–66.7% to 89.5%–96.6%. Both assays specifically detected the target viruses without cross‐reacting to SVV‐1 or to other common porcine viruses. An 18S rRNA housekeeping gene that was amplified from multiple cloven‐hoofed animal species was used as an internal control. The prevalence study did not detect any FMDV, but SVV‐1 was detected from multiple types of swine samples with an overall positive rate of 10.5% for non‐serum samples.

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“…Co więcej, zastosowanie wielu specyficznych sond molekularnych w pojedynczej reakcji typu real-time PCR pozwoliło na jednoczesną identyfikację nawet 12 patogenów, włączając m.in. : ASFV, CSFV, FMDV, PRRSV czy cirkowirus świń typu 2 -PCV2 (35). Należy zwrócić uwagę, iż wiele z metod diagnostycznych zalecanych przez OIE opiera się na technice real-time PCR, która jest obecnie jedną z najbardziej wiarygodnych, czułych i specyficznych metod diagnostycznych (38).…”

Section: Artykuł Przeglądowy Reviewunclassified

Increasing importance of genomics in recognition of infectious animal diseases taking into account OIE requirements

Grzegorz.¹,

Truszczyński²,

Zygmunt³

2017

Medycyna Weterynaryjna

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Currently, the diagnosis methods applied in bacteriology and virology are frequently focused at modern molecular methods such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP) or confirmatory methods for already identified infectious agents of animals such as high throughput sequencing- HTS, or next-generation sequencing (NGS). A number of these methods are officially recommended by the World Organisation for Animal Health (OIE) for the routine diagnosis of major animal diseases with the greatest economical impact. The aim of this paper is to provide to a reader an overview on recent genomic methods used in diagnosis of infectious animal diseases along with their advantages and limitations. These methods facilitate efficient and reliable identification of animal pathogens and allow to characterize and sometimes to identify a newly recognized species of bacteria and viruses.

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A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses

Cited by 31 publications

References 32 publications

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

Development and evaluation of multiplex real‐time RT‐PCR assays for the detection and differentiation of foot‐and‐mouth disease virus and Seneca Valley virus 1

Increasing importance of genomics in recognition of infectious animal diseases taking into account OIE requirements

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