PCR-DGGE技术用于猪场沼气池细菌群落分析的条件优化

Optimization of Denaturing Gradient Gel Electrophoresis for Bacterial Communities in Biogas Slurry of Pig Farm

本研究采用变性梯度凝胶电泳(PCR-DGGE)技术研究猪场沼气池细菌群落,对基于16S rDNAV3区的PCR-DGGE电泳的最佳变性剂梯度范围、电泳时间、染色时间进行优化。研究结果表明,最佳变性剂梯度范围为35%～60%,电泳时间为12h,SYBR Green Fluorescent Dye染料的染色时间是30min,优化后的PCR-DGGE确保了实验的准确性、灵敏度和可重复性。运用此优化后的PCR-DGGE技术对5个猪场沼液细菌群落进行了研究,获得了较丰富的多样性。该实验为深入研究畜禽养殖粪污治理的菌种筛选奠定了基础。

Ploymerase chain reaction and denaturing gredient gel electrophoresis（PCR-DGGE） technique was used to reveal the bacterial community composition and diversity associated with biogas slurry of pig farm.In order to improve the accuracy,sensitivity and repeatability of DGGE based on 16S rDNA V3 region,the running parameters of DGGE were optimized by level electrophoresis,electrophoresis time and dying time,respectively.The results showed that the best range of denaturing gradients was 35%~60% in 8% polyacrylamide gels,running time was 12 hours,and the dying time was 30 min using SYBR Green Fluorescent Dye.Furthermore,bacterial community from 5 biogas digesters of pig farms was also performed by using these optimized reaction conditions.It was found that the 5 samples exhibited effective separation and rich microbial diversity.This indicated that different samples would result in the distinct change of microbial diversity and microbial community structure of biogas slurry.PCR-DGGE was proven to be a powerful tool for describing the bacterial flora in biogas slurry of pig farms.The results provide a basis for studying the bacterial community in livestock manure treatment.