IHHNV PCR检测方法改进及广西流行株基因型分析

Improvement of PCR detection method for IHHNV and genotypes analysis of epidemic strains in Guangxi

[目的]建立并优化传染性皮下及造血组织坏死病毒(IHHNV)检测及其基因分型的套式PCR,并确定IHH-NV在广西流行的基因型,为有效防控广西对虾传染性皮下及造血组织坏死病(IHHN)提供参考依据。[方法]在以PCR检测IHHNV现有标准的基础上,于389F/R和309F/R两对引物扩增片段之外的绝对保守区域设计外引物IHHNV-WF/WR,优化退火温度和引物浓度后建立IHHNV检测及基因分型的套式PCR,并通过与一步法PCR对比以验证其优越性;采用建立的套式PCR对546株2010 2018年收集的IHHNV广西流行株进行基因型分析,确定IHHNV在广西流行的基因型。[结果]优化后的第一轮PCR反应体系20.0 L:2×F8 FastLong PCR MasterMix 10.0 μL,上、下游引物(20 μmol/L)各0.8 μL,DNA模板2.0 μL,无核菌酶灭菌水补足至20.0 μL。扩增程序:94.0 ℃预变性3 min;94.0 ℃ 10 s,59.0 ℃ 15 s,72.0 ℃ 15 s,进行35个循环;72.0 ℃延伸5 min。套式PCR检测IHHNV的灵敏度较一步法PCR提高100倍;对100份IHHNV阳性临床样品的检测结果与一步法PCR的检测结果一致,符合率达100%。从546株IHHNV广西流行株中均能扩增获得309 bp的目的条带,全部为感染型(基因1型和基因2型)。[结论]在PCR检测IHHNV现有标准基础上建立的IHHNV检测及基因分型套式PCR,其灵敏度较一步法PCR提高100倍,尤其适用于检测IHHNV含量低的样品,可为疫病监测及进出口检疫提供更敏感的技术手段。当前广西流行的IHHNV均为感染型(基因1型和基因2型)。

【Objective】The nested PCR detection methods were developed in order to provide technical support for detection and genotypes analysis of infectious hypodermal and hematopoietic necrosis virus(IHHNV),and the genotypes of IHHNV epidemic in Guangxi were investigated to offer a reference for effective prevention and control of infectious subcutaneous and hematopoietic necrosis virus(IHHN)for prawn in Guangxi.【Method】A pair of outer primers named IHHNV- WF/WR were designed on the conserved domain which located at both ends of primers 389F/R and 309F/R used in the present standards for PCR detection of IHHNV. Then the nested PCR detection methods were developed following optimization of annealing temperature and primers concentration. The superiority of the method was evaluated by comparing with former one-step PCR. A total of 546 IHHNV strains from Guangxi during 2010-2018 were conducted genotypes analysis using the nested PCR. The epidemic strains of IHHNV in Guangxi were identified.【Result】The optimized first-round PCR amplification reactions 20.0 μL included 10.0 μL of 2×F8 FastLong PCR Master Mix,0.8 μL of upstream and downstream primers(20 μmol/L),2.0 μL of the DNA template and sterile water to a final volume of 20.0 μL. PCR amplification was carried out as follows:3 min initial denaturation step at 94 ℃;followed by 35 cycles of 94 ℃ for 10 s,59 ℃ for 15 s and 72 ℃ for 15 s;with a final extension step of 5 min at 72 ℃. The nested PCR methods developed were 100 times more sensitive than former one-step PCR methods. The nested PCR was used to detect 100 IHHNV-positive clinical samples, the results were consistent with former one-step PCR,coincidence rate was 100%. All 546 IHHNV epidemic strains from Guangxi could amplify 309 bp target bands,and they were infectious genotypes(genotype 1 and genotype 2).【Conclusion 】The nested PCR methods developed in this study on the current basis for detection and genotypes analysis of IHHNV are 100 times more sensitive than former one-step PCR methods,and can provide more sensitive means for detecting IHHNV in surveillance and quarantine samples which contain small amount of IHHNV. IHHNV epidemic strains in Guangxi are all infectious genotypes(genotype 1 and genotype 2)at present.