PCR检测对虾白斑综合征病毒(WSSV)中假阴性的探讨

PCR FALSE NEGATIVE RESULT IN WHITE SPOT SYNDROME VIRUS(WSSV) DETECTION

采用PCR、核酸探针斑点杂交、组织切片等方法研究了PCR检测白斑综合征病毒(WSSV)中的假阴性问题。设计了一对引物用于PCR检测WSSV,PCR扩增的产物长度为235bp,检出极限为0.1pg WSSV DNA。结果表明,PCR检测15例攻毒螯虾鳃样品时出现一例阴性,而经10—106倍稀释后的样品却呈现阳性,推断PCR出现了假阴性。核酸探针斑点杂交及组织切片结果表明注射WSSV的螯虾鳃组织确已被严重感染,进一步证实了PCR假阴性。根据PCR的检出极限及模板稀释梯度,推算出该PCR能成功扩增的引物与模板浓度的比例范围约为2.4×105—2.4×1010。

Based on whole DNA sequence of white spot syndrome virus （WSSV）, a pair of primer was designed for the detection of WSSV with Primer Premier 5.0 program. The primer pair was 5＇-CCAAGA- CATACTAGCGGATA-3＇ and 5＇-GACGACCCTGACAGAATTAC-3＇ with a product fragment of 235bp, whose PCR detection limit was 0.1pg WSSV DNA. 15 Procamburas clarkii were injected with WSSV inoculum. One side gill of dead P clarkii was fixed with SEMP to extract DNA for PCR and probe dot blot hybridization. For the samples with negative PCR detection result, the other side gill was fixed with Davidson＇s solution for routine paraffin section. A negative result occurred in all 15 WSSV inoculated P clarkii in PCR detection. The DNA of the negative samples was then diluted 10-fold consecutively from 10 to 10^9 times for PCR. The detection showed that positive results occurred when DNA sample of PCR negative was diluted to 10- 10^6 times. No anticipated product appeared in the samples diluted to 10^7- 10^9 times because the templates were too rare. Since all the positive and negative controls were normal, we believed that false PCR negative result occurred in undiluted sample DNA. In DNA probe dot blot hybridization detection, DNA of PCR-negative sample and its 10× and 10^2×- time diluted solutions were all positive. Inflate nuclei, destructive and necrotic tissue were observed in P clarkii gill tissue section. Results of DNA probe dot blot hybridization detection and histopathological section showed that P. clarkii gill tissue was seriously infected by WSSV, which proved again that the negative result of PCR was false. Since the templates that diluted 10- 10^6 times was positive in PCR, the false negative result was probably resulted from too many templates that reduced the ratio of primer to template and lessened the PCR. Based on PCR detection limit and template dilution times, the best ratio of primer to template for successful amplification was 2.4× 10^5- 2.4× 10^10. The false negative ratio was 6.6% for 1 false negative result in 15 WSSV injected P. clarkii. The ratio would be higher if the template was purified WSSV or WSSV DNA. So the PCR false negative result should not be ignored and the application of PCR technique in WSSV detection should be considered. PCR nega- tive result arisen from excessive templates appeared only in extreme situation such as artificial infection or dead shrimp in explosive WSSV pond, which is very unlikely occur for the samples with low WSSV content. PCR false negative result arisen from excessive templates can be avoid by template dilution. A reliable way to check PCR false negative result was to use another detection method at a time.