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用已报道的植原体通用引物对葡萄黄化病和枣疯病植原体进行常规PCR及巢式PCR检测,并对PCR反应体系进行优化。结果表明:常规PCR扩增出了1.5kb的特异片段,所需PCR模板DNA用量为20ng/μL;在PCR基础上的巢式PCR扩增出1.2kb的特异片段,所需PCR模板DNA用量为2pg/μL。检测灵敏度提高约10000倍。优化试验表明:25μL反应体系中,MgCl2用量对扩增效果影响最大。最终最佳反应体系确立为:25mM MgCl21.7μL,2.5mM dNTP1.8μL,5U/μL Taq酶可选用0.2μL,10mM引物对各0.2μL,最佳退火温度为45.0℃。
The common primers of phytoplasma were used to detect the yellowing disease and the jujube baker’s phytoplasma by routine PCR and nested PCR, and the PCR reaction system was optimized. The results showed that: 1.5kb specific fragment was amplified by conventional PCR, the required amount of PCR template DNA was 20ng / μL; on the basis of PCR, a 1.2kb specific fragment was amplified by nested PCR, the amount of PCR template DNA needed was 2pg / μL. Detection sensitivity increased by about 10000 times. Optimization experiments show that: 25μL reaction system, the amount of MgCl2 greatest impact on the amplification effect. The final optimal reaction system was established as follows: 21.7 μL of 25 mM MgCl 2, 1.8 μL of 2.5 mM dNTP, 0.2 μL of 5 μL / μL Taqzyme and 0.2 μL each of 10 mM primer. The optimal annealing temperature was 45.0 ° C.