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期 刊 ：植物保护学报 2007年

关键词：柑桔黄龙病；常规PCR；半巢式PCR；巢式PCR；灵敏度；

Keywords:citrus huanglongbing, PCR, semi-nested PCR, nested PCR, detection sensitivity,

摘 要 ：对目前常用的5对引物 (fOI1/rOI2c、fOI2/r23S1、fA2/rJ5、fP₅₃₅/rP₅₃₅、fP₄₀₀/rP₄₀₀) 检测柑桔黄龙病菌的灵敏度进行了比较。结果发现,不同引物对的检测灵敏度不同,5对引物检测灵敏度由高到低依次为:fP₄₀₀/rP₄₀₀>fP₅₃₅/rP₅₃₅>fA2/rJ5>fOI1/rOI2c>fOI2/r23S1;引物对 fP₄₀₀/rP₄₀₀比广谱引物对fOI2/r23S1的灵敏度高近千倍。半巢式PCR及巢式PCR的检测灵敏度远高于常规PCR,可检测出接近极限浓度10^-7ng/μL,二者没有明显的差别。因此,按照不同目的选择合适的检测引物非常重要,特别是当待测样品中的黄龙病菌浓度极低时,应选择高灵敏度的小片段特异性引物对。

Abstract:A comparison of the detection sensitivity of five primer pairs (fOI1/rOI2c, fOI2/r23S1, fA2/rJ5, fP₅₃₅/rP₅₃₅, fP₄₀₀/rP₄₀₀) for citrus huanglongbing pathogen was carried out using simple PCR, semi-nested PCR and nested PCR. The result showed that different primer pairs had distinct detection sensitivity. The most sensitive primer pair fP₄₀₀/rP₄₀₀ was nearly thousand times more sensitive than primer pair fOI2/r23S1, from the highest to the lowest were fP₄₀₀/rP₄₀₀>fP₅₃₅/rP₅₃₅>fA2/rJ5>fOI1/rOI2c>fOI2/r23S1. The sensitivity of semi-nested PCR and nested PCR were much higher than that of simple PCR, which can detect out the utmost concentration of 10^-7ng/μL, and theres no obvious difference between each other. Therefore, it is important to choose sensitive primer pairs which amplify much smaller target fragment for the detection of citrus huanglongbing pathogen. Especially when citrus huanglongbing pathogen is extremely low in the samples, it is essential to choose much more sensitive primes in order to avoid false negative.

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