Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection

Objectives: Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods: Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results: Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance: These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection

Evaluation of a Field-Deployable Reverse Transcription-Insulated Isothermal PCR for Rapid and Sensitive On-Site Detection of Zika Virus

Background: The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources. Methods: We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively. Results: These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/μl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83). Conclusions: The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas

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

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 50 untranslated region (50 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 RTPCR (RT-qPCR) along with the AGID test. Methods: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 50 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.Instituto de VirologíaFil: Cook, R.F. Louisiana State University. School of Veterinary Medicine. Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences; Estados UnidosFil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria. Cátedra de Enfermedades Infecciosas; ArgentinaFil: Lee, Pei-Yu Alison. GeneReach USA, Lexington; Estados UnidosFil: Tsai, Chuan-Fu. GeneReach USA, Lexington; Estados UnidosFil: Shen, Yu-Han. GeneReach USA, Lexington; Estados UnidosFil: Tsai, Yun-Long. GeneReach USA, Lexington; Estados UnidosFil: Chang, Hsiao-Fen G. GeneReach USA, Lexington; Estados UnidosFil: Wang, Hwa-Tang Thomas. GeneReach USA, Lexington; Estados UnidosFil: Balasuriya, Udeni B.R. Louisiana State University. School of Veterinary Medicine. Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences; Estados Unido

Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus

Abstract Background The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources. Methods We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively. Results These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/μl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83). Conclusions The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas