Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device

Kuo, Hung-Chih; Lo, Dan-Yuan; Chen, Chiou-Lin; Tsai, Yun-Long; Ping, Jia-Fong; Lee, Chien-Hsien; Lee, Pei-Yu; Chang, Hsiao-Fen Grace

doi:10.3382/ps/pew228

Cited by 14 publications

(10 citation statements)

References 32 publications

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“…The iiPCR technology and a commercially available field-deployable device (POCKIT™ combo system), which includes a taco™ mini Automatic Nucleic Acid Extraction System and a POCKIT™ Nucleic Acid Analyzer (GeneReach USA, Lexington, MA, USA), allow automatic detection and interpretation of PCR results within 1-1.5 h [36]. It has been shown that iiPCR or RT-iiPCR assays have excellent sensitivity and specificity for the detection of various targets, including swine pathogens, such as classical swine fever virus (CSFV), FMDV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV) [38][39][40][41], and various pathogens in shrimp, dogs, cats, poultry, ruminants, and horses [42][43][44][45][46][47][48][49][50][51][52].…”

Section: Introductionmentioning

confidence: 99%

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

et al. 2019

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Background Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. Results Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59–100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39–100%, κ = 0.97). The two samples with discrepant results had relatively high C T values. Conclusions The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.

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Section: Introductionmentioning

confidence: 99%

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

et al. 2019

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“…The equipment including an automated nucleic acid extraction system (taco TM mini) required for iiPCR (POCKIT TM Nucleic Acid Analyzer a , POCKIT TM ) is specifically designed with the option of using batteries as an electrical power source and lyophilised reagents avoiding the need for a cold chain to be fully field-deployable [26]. This enables it to be used for point of need diagnosis plus results are obtainable within 1 h. Moreover, a number of assays have been successfully developed for detection of pathogens associated with humans, companion animals, livestock and farmed aquatic species based on the iiPCR/POCKIT TM system and all have been shown to have equivalent specificity and sensitivity to RT-qPCR [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. In this report, an EIAV RT-iiPCR based on the POCKIT TM system designed to aid in the diagnosis of EIAV-infected equids was evaluated by comparison with a conventional EIAV RT-qPCR assay, where both assays targeted the same region of the viral genome although not using identical primers and probes.…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT‐PCR assay to aid diagnosis under field conditions

Cook

Barrandeguy

Lee

et al. 2018

Equine Veterinary Journal

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Summary Background Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti‐EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. Objectives To evaluate a field‐deployable, reverse transcription‐insulated isothermal PCR (RT‐iiPCR) assay targeting the conserved 5′ untranslated region (5′ UTR)/exon 1 of the tat gene of EIAV. Study design The analytical and clinical performance of the newly developed EIAV RT‐iiPCR was evaluated by comparison with a EIAV real‐time RT‐PCR (RT‐qPCR) along with the AGID test. Methods Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 5′ UTR/tat gene and samples from two EIAV‐positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT‐qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. Results EIAV RT‐iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT‐qPCR. However, the RT‐qPCR and RT‐iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. Main limitations Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT‐PCR‐based tests. Conclusions Although EIAV RT‐iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or “serologically silent” equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field‐deployable design enable utilisation of EIAV RT‐iiPCR even in remote regions.

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“…The POCKIT iiPCR system provides an efficient and cost‐effective alternative for mass testing of high‐risk dogs for B. gibsoni . Although this study focused on B. gibsoni , the field‐deployable iiPCR platform has potential to aid in rapid detection of a variety of animal and human infections under field conditions …”

Section: Discussionmentioning

confidence: 99%

Rapid Diagnosis of Babesia gibsoni by Point‐of‐Need Testing by Insulated Isothermal PCR in Dogs at High Risk of Infection

Cooke

Frenzer

Tucker

et al. 2018

Veterinary Internal Medicne

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BackgroundDogs seized by law enforcement agencies during dogfighting investigations are at increased risk of Babesia gibsoni infection. A rapid and cost‐effective diagnostic test would increase the feasibility of mass screening of dogs for infection and monitoring treatment efficacy in B. gibsoni‐infected dogs.ObjectiveTo determine the performance of a point‐of‐need insulated isothermal PCR (iiPCR) test for diagnosis of B. gibsoni in dogs rescued in dogfighting investigations.AnimalsTwo hundred and thirty‐three dogs seized in dogfighting investigations.MethodsCross‐sectional study. Whole blood samples were tested for B. gibsoni and Babesia spp. by iiPCR. Results were compared to a reference standard comprised of concordant results from real‐time PCR in a commercial diagnostic laboratory and antibody titers.ResultsThe iiPCR system was quick to learn, portable, and had a short processing time of <2 hours. Sensitivity and specificity of the iiPCR assay for B. gibsoni were 90% (95% confidence interval [CI] 81–95%) and 99% (CI, 95–100%), respectively. Sensitivity and specificity of the iiPCR assay for Babesia spp. were 87% (CI, 78–93%) and 98% (CI, 0.94–99%), respectively.Conclusions and Clinical ImportanceThe iiPCR system produced few false‐positive results, indicating that positive results are likely to represent true infections when used in high‐risk animals. The iiPCR system can fail to identify 10–15% of truly infected dogs. However, the portability, speed, and economy of the iiPCR system compared to testing through a reference laboratory can allow rescue groups to screen and identify infection in more dogs.

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Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device

Cited by 14 publications

References 32 publications

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT‐PCR assay to aid diagnosis under field conditions

Rapid Diagnosis of Babesia gibsoni by Point‐of‐Need Testing by Insulated Isothermal PCR in Dogs at High Risk of Infection

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