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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。
The PCR detection of nucleic acid probe spot blotting and tissue biopsy was used to study the false negative detection of white spot syndrome virus (WSSV) by PCR. A pair of primers was designed for PCR detection of WSSV. The length of the product amplified by PCR was 235bp and the detection limit was 0.1pg WSSV DNA. The results showed that one negative case was found when PCR was performed on gill samples of 15 chewed shrimp while the 10-106-fold diluted samples showed positive results. The PCR was false negative. Nucleic acid probe dot blotting and biopsy results showed that WSSV injected crayfish gill tissue has indeed been severely infected, further confirming the PCR false negative. According to the detection limit of PCR and the gradient of template dilution, the ratio of primer to template concentration of PCR amplification was estimated to be about 2.4 × 105-2.4 × 1010.