Wang Jianchang, Liu Libing, Wang Jinfeng, Sun Xiaoxia, Yuan Wanzhe
Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau, Xinhua District, Shijiazhuang, Hebei, China.
College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, China.
PLoS One. 2017 Jan 3;12(1):e0166903. doi: 10.1371/journal.pone.0166903. eCollection 2017.
Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.
猫疱疹病毒1型（FHV-1）是一种有包膜的双链DNA病毒，是猫上呼吸道疾病（URTD）和眼部疾病的主要病原体之一。目前，聚合酶链反应（PCR）仍然是FHV-1感染的金标准诊断工具，但相对昂贵，需要设备完善的实验室，且不适合现场检测。重组酶聚合酶扩增（RPA）是一种等温基因扩增技术，已被用于传染病的分子诊断研究。在本研究中，开发并验证了一种用于检测FHV-1的外切核酸酶RPA检测方法。设计了特异性靶向FHV-1胸苷激酶（TK）基因的引物。RPA反应在39℃成功进行，20分钟内获得结果。以含有TK基因的不同拷贝数的重组质粒DNA为模板，我们发现外切核酸酶RPA的检测限为102个DNA拷贝/反应，与实时PCR相同。外切核酸酶RPA检测方法未交叉检测猫泛白细胞减少症病毒、猫杯状病毒、牛疱疹病毒1型、伪狂犬病病毒或鹦鹉热衣原体（猫URTD中的一组重要病原体）或α疱疹病毒亚科中的其他病毒，显示出高特异性。通过检测120份猫的鼻拭子和眼结膜拭子对该检测方法进行了验证，并将结果与实时PCR的结果进行了比较。两种检测方法在临床样本中提供了相同的检测结果。与实时PCR相比，外切核酸酶RPA检测方法使用的设备更简单、便于携带，反应完成得更快。此外，真空密封袋中的商业RPA试剂可在室温下耐受数天而不失活，适合现场检测的运输和储存。综上所述，外切核酸酶RPA检测方法是一种简单、快速且经济高效的实时PCR替代方法，适用于不太先进的实验室和FHV-1感染的现场检测。
