嵌套引物P1/P7-U3/U5检测葡萄黄化(stolbur)植原体时扩增出的额外片段分析

 当采用嵌套引物P1/P7(U5/P7) U3/U5的巢式PCR检测植原体时,伴随着正常的0.85kb片段还经常扩增出一条额外片段。与其它常见的非特异性片段不同,该额外片段十分稳定,与正常片段总是同步出现,二者的强度呈正比。经测定,额外片段的大小为0.36kb。迄今,这一现象仅在采用P1/P7(U5/P7)-U3/U5引物的巢式PCR中出现。在采用P1/P7、U5/P7或-U3/U5引物的常规PCR中从未出现过。另外,已知这一现象至少在葡萄黄化stolbur和榆黄化植原体的检测过程中出现。对该现象产生的原因进行了深入研究,方法是将额外片段从凝胶中分离出来,用不同的引物在不同的条件下进行重新扩增,同时结合已知的植原体16SrRNA基因序列进行综合分析判断。结果表明,该额外片段源于植原体16SrRNA基因上存在的一个与U5引物部分互补的位点。对额外片段的测序结果进一步证实了分析的正确性。据此,指出了该额外片段在植原体检测中的可能用途。

Analysis of an extra fragment amplified by P1/P7 nested U3/U5 primers in grapevine yellows (stolbur)phytoplasma detection

The 16S rRNA gene-based PCR has been developed as a routine method for phytoplasma detection. When P1/P7 (or U5/P7) nested U3/U5 primers were used in PCR,the normal 0.85 kb product band was often accompanied by an unexpected 0.36 kb fragment. The occurrence and intensity of this extra band was tightly related to the normal band. This phenomenon was only observed in the nested-PCR detection using P1/P7 (or U5/P7) nested U3/U5 primers,and never occurred in routine PCR using P1/P7,U5/P7 or U3/U5 primer pairs. So far,the extra PCR band was found at least in grapevine yellows (stolbur) and elm yellows phytoplasma detection. Further PCR amplification and nucleotide analysis revealed that this extra fragment located on phytoplasma 16S rRNA gene with a partial complement site of U5 primer. These results revealed the potential usage of this extra band in phytoplasma detection.