Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer

Balasuriya, Udeni B. R.; Lee, Pei-Yu; Tiwari, Akash; Skillman, Ashley; Nam, Bora; Chambers, Thomas M.; Tsai, Yun-Long; Ma, Lijuan; Yang, Pan‐Chyr; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

doi:10.1016/j.jviromet.2014.06.016

Cited by 40 publications

(29 citation statements)

References 24 publications

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“…Thus, the simplicity and efficiency of the RT- iiPCR coupled with the POCKIT nucleic acid analyzer (RT-iiPCR/ POCKIT) to rapidly amplify nucleic acids under isothermal conditions suggested that this assay could be a potential alternative for detection of DENV especially in remote field settings. This system has been shown to offer sensitivity, specificity, and clinical performance comparable to those of reference real-time PCR or nested PCR methods for various microbial pathogens in various clinical sample types (36)(37)(38)(39)(40)(41)(42)(43)(44)(45). One of the major advantages of RT-iiPCR is its simple protocol.…”

Section: Discussionmentioning

confidence: 99%

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

Rajapakse

Kularatne

et al. 2016

J Clin Microbiol

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e Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3= untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n ‫؍‬ 220) and individuals not suspected of dengue virus infection (n ‫؍‬ 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries. Dengue virus (DENV) is a mosquito-borne human pathogen that belongs to the genus Flavivirus in the family Flaviviridae (1). DENV is an enveloped virus with a single-stranded, positivesense RNA genome approximately 10.7 kb in length (2). The genomic RNA includes 5= and 3= untranslated regions (UTRs) and a single open reading frame that encodes a single polyprotein that is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3). DENV is comprised of four distinct serotypes (DENV-1, -2, -3, and -4), and all four serotypes are currently circulating in the Pacific Islands, Americas, Asia, and Africa. In addition to this, distinct genotypes have been identified within each serotype (4, 5), highlighting the extensive genetic variability of the DENV serotypes.DENV infection is considered a major public health problem in developing tropical countries where the virus is endemic, and it is continuously spreading to new geographical areas around the world (6, 7). Moreover, frequent international travel to regions where dengue is endemic or epidemic increases the risk of DENV infection for travelers (8,9), and travel contributes to the escalating numbers of imported dengue cases in temperate regions. More than 2.5 billion people ...

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Section: Discussionmentioning

confidence: 99%

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

Rajapakse

Kularatne

et al. 2016

J Clin Microbiol

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“…The downscaling in size of nucleic acid extraction and high‐speed real‐time PCR platforms is opening new opportunities for utility at or near the source of collection . In many cases, advancements in developing rapid, point‐of‐capture real‐time PCR platforms have been pioneered for veterinary applications, facilitating diagnosis and treatment in companion animal medicine as well as management and control of infectious disease affecting livestock production …”

Section: The Potential Of Rapid Point‐of‐capture (Poc) Diagnosticsmentioning

confidence: 99%

New frontiers in applied veterinary point‐of‐capture diagnostics: Toward early detection and control of zoonotic influenza

Schar

Padungtod

Nguyen

et al. 2019

Influenza Resp Viruses

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Among the chief limitations in achieving early detection and control of animal‐origin influenza of pandemic potential in high‐risk livestock populations is the existing lag time between sample collection and diagnostic result. Advances in molecular diagnostics are permitting deployment of affordable, rapid, highly sensitive, and specific point‐of‐capture assays, providing opportunities for targeted surveillance driving containment strategies with potentially compelling returns on investment. Interrupting disease transmission at source holds promise of disrupting cycles of animal‐origin influenza incursion to endemicity and limiting impact on animal production, food security, and public health. Adoption of new point‐of‐capture diagnostics should be undertaken in the context of promoting robust veterinary services systems and parallel support for operationalizing pre‐authorized plans and communication strategies that will ensure that the full potential of these new platforms is realized.

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“…The reaction worked on a portable POCKIT™ Nucleic Acid Analyzer (POCKIT™; GeneReach, Taichung, Taiwan), which detected and processed reaction signals automatically to generate easy final positive and negative readouts. Carried out in a capillary tube and driven by the Rayleigh-Bènard convention (Krishnan et al., 2002), the reaction could generate detectable signals in less than 1 h. The iiPCR/POCKIT™ system has been demonstrated to achieve specific and sensitive detection of several bacterial and viral infections of veterinary interest in companion animals, livestock animals, and aquaculture animals (Tsai et al., 2012; Tsen et al., 2013; Balasuriya et al., 2014; Tsai et al., 2014; Wilkes et al., 2014; Ambagala et al., 2015; Lung et al., 2015; Wilkes et al., 2015a,b). In addition, a nucleic acid extraction step is generally required before PCR to remove reaction inhibitors in the sample matrix (Schrader et al., 2012).…”

Section: Introductionmentioning

confidence: 99%

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

Kuo

Chen

et al. 2017

Poultry Science

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Mycoplasma synoviae (MS), causing respiratory diseases, arthritis, and eggshell apex abnormalities in avian species, is an important pathogen in the poultry industry. Implementation of a biosecurity plan is important in MS infection management. Working on a field-deployable POCKIT™ device, an insulated isothermal polymerase chain reaction (iiPCR) assay has a potential for timely MS detection on the farm. The MS iiPCR assay had limit of detection 95% of about 9 genome equivalents by testing serial dilutions of a standard DNA. The detection endpoint of the assay for detection of MS genomic DNA was comparable to a reference real-time PCR. The assay did not crossreact with other important avian pathogens, including avian reovirus, Mycoplasma gallisepticum, Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Salmonella Pullorum. When 92 synovial fluid and respiratory tract swab samples collected from chickens, turkeys, and geese suspected of MS infection were tested, the clinical performance of the MS iiPCR had 97.8% agreement (Cohen's kappa value, 0.95) with that of the reference real-time PCR. In conclusion, the MS iiPCR/POCKIT™ system, working with field-deployable manual or automatic nucleic acid extraction methods, has potential to serve as a rapid and sensitive on-site tool to facilitate timely detection of MS.

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Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer

Cited by 40 publications

References 24 publications

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

New frontiers in applied veterinary point‐of‐capture diagnostics: Toward early detection and control of zoonotic influenza

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

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