乌梁素海沉积物中厌氧氨氧化细菌PCR扩增方案优化

Optimization of PCR amplification project for anammox bacteria in Wuliangsuhai sediments

[目的]对乌梁素海沉积物中厌氧氨氧化细菌的PCR扩增方案进行优化。[方法]采集乌梁素海沉积物样品,提取细菌总DNA,以厌氧氨氧化细菌功能基因Hzo基因为研究对象,通过基于普通PCR和巢式PCR筛选适宜的引物、探究不同的PCR退火条件,确立PCR引物的最佳选择以及最佳扩增条件。[结果]以HzoAB4F/HzoAB4R为引物,PCR退火温度为53℃的条件下,能获得预期的600 bp左右的条带,但由于引物二聚体的出现,对于后续菌群结构的研究可能带来一些影响,因此该扩增策略还需要继续完善;其他引物进行的PCR反应扩增,获得的结果特异性均较差。巢式PCR方案(HzoAB1F/HzoAB1R为第一轮引物、HzoAB4F/HzoAB4R作为第二轮引物),PCR退火温度为55℃的条件下,可大大提高对厌氧氨氧化细菌功能基因Hzo扩增的特异性。[结论]成功对乌梁素海沉积物中厌氧氨氧化细菌功能基因Hzo进行扩增,研究发现巢式PCR方案适用于环境中厌氧氨氧化细菌的分子生态学研究。

[Objective]The PCR amplification project for anammox bacteria in Wuliangsuhai sediments was optimized.[Method]The sediment samples from Wuliangsuhai wetland were collected to extract total DNA,and a functional gene of anammox bacteria,Hzo gene,was used as the research object.Through selecting suitable primers and exploring different PCR annealing conditions,the best PCR primer selection and amplification conditions were established.[Result]With HzoAB4 F/HzoAB4 R as the primers and the annealing temperature of PCR at 53℃,the expected bands could be obtained.However,due to the emergence of primer dimer,the amplification strategy needs to be improved;the results obtained by the PCR reaction with other primers were poor.Nested PCR(HzoAB1 F/HzoAB1 R as the first round primers,HzoAB4 F/HzoAB4 R as the second round primers)and the annealing temperature at 55℃,which can obtain the satisfactory PCR results of anammox bacteria.[Conclusion]The functional gene of anammox bacteria from Wuliangsuhai sediments was successfully amplificated.Our data suggest that Nested PCR project is suitable for the study of molecular ecology of anammox bacteria in the environment.