Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer

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• 作者：Udeni B.R. Balasuriya^a ; ^{ubalasuriya@uky.edu} ; Pei-Yu Alison Lee^b ; ^{peiyu329@genereachbiotech.com} ; Yun-Long Tsai^b ; ^{fiwind.tsai@genereachbiotech.com} ; Chuan-Fu Tsai^b ; ^{marktsai@genereachbiotech.com} ; Yu-Han Shen^b ; ^{yuki7510zz@genereachbiotech.com} ; Hsiao-Fen Grace Chang^b ; ^{gracechang@genereachbiotech.com} ; Ashley Skillman^a ; ^{ashley.skillman@uky.edu} ; Hwa-Tang Thomas Wang^b ; ^{thomaswang@genereachbiotech.com} ; Sté ; phane Pronost^c ; ^{Stephane.PRONOST@laboratoire-labeo.fr} ; Yan Zhang^d ; ^{YZhang@agri.ohio.gov}
• 关键词：Equine herpesvirus-1 ; Equine herpesvirus myeloencephalopathy ; EHM ; iiPCR
• 刊名：Journal of Virological Methods
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：241
• 期：Complete
• 页码：58-63
• 全文大小：426 K
• 卷排序：241

文摘

Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48–99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.