Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, 889-2192 Miyazaki, Japan.
J Virol Methods. 2009 Dec;162(1-2):81-7. doi: 10.1016/j.jviromet.2009.07.018. Epub 2009 Jul 29.
A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture.
一种实时逆转录环介导等温扩增（real-time RT-LAMP）方法被应用于检测虾类黄头病毒（YHV）的复制酶多蛋白编码基因。这是一种新颖的、针对基因的检测方法，在等温条件下使用一组六个专门设计的引物，可特异性地识别靶基因的八个独特序列，从而对核酸进行高特异性、高灵敏度和快速扩增。该方法甚至可以检测到低拷贝数的 DNA，并且基于镁焦磷酸浊度检测，通过廉价的光度计进行定量分析。我们制定了一个用户友好的协议，最佳条件标准化为 63°C 60 分钟。使用该方案，检测灵敏度比广泛使用的 YHV 巢式 RT-PCR 系统高 10 倍。使用其他虾类病毒 DNA/cDNA 和 YHV 阴性虾进行交叉反应分析表明，该检测方法具有高度特异性。还使用实时 RT-LAMP 方法对内部对照基因 EF-1alpha 进行检测，以比较感染虾不同器官中 YHV 基因的表达情况，所得标准曲线显示出高相关系数值。这些结果表明，该检测方法可广泛应用于追求无 YHV 虾类养殖的新型定量检测方法。
