Method for obtaining nucleotide sequence in V3 region of 16S rRNA genes of bacteria and special primer thereof

Abstract

The invention discloses a method for obtaining a nucleotide sequence in the V3 region of 16S rRNA genes of bacteria and a special primer thereof. The special primer is a specific primer pair consisting of nucleotides having a sequence 17 and a sequence 19 in a sequence table. The method comprises the following steps of: (1) carrying out PCR amplification by using a universal primer and using the genome DNA of the bacterium as a template, wherein the universal primer is a primer pair consisting of nucleotides having a sequence 17 and a sequence 18 in the sequence table; (2) carrying out PCR amplification by using the specific primer and using the PCR product of the step (1) as a temple; (3) sequencing the PCR product in the step (2) by using a sequencing primer, wherein the nucleotide sequence of the sequencing primer is shown as the sequence 20 in the sequence table. The primer pair aiming at the V3 region of 16S rRNA genes for bacterial diversity analysis provided by the invention can be directly used for sequencing without carriers, which is beneficial to reducing the experimental cost, shortening the experimental period and enhancing the accuracy of the experiment. The specific primer pair and the method are particularly suitable for bacterial diversity test of environmental sample aiming at the V3 region of 16S rRNA genes by using PCR-DGGE (PCR-Denaturing Gradient Gel Electrophoresis) technology.

Description

technical field [0001] The invention relates to a method for obtaining the nucleotide sequence of the V3 region of the bacterial 16S rRNA gene and special primers thereof. Background technique [0002] Bacterial ribosomal nucleotide genes usually contain nine (V1-V9) regions. However, among these nine regions, there are also differences in the length of the variable region and the diversity of the nucleotide sequence of the variable region. Among them, the base sequence diversity of V3 and V6 regions was the highest. The V6 region is relatively short, so the V3 region (about 200bp) in the 16S rRNA gene has become one of the most commonly used molecular markers. The PCR-DGGE technique and the extended PCR-TGGE are widely used in various fields of microbial molecular ecology research. Because of the large amount of information, almost all genera of bacteria can be distinguished and the sequence length is within the best separation effect of DGGE gel, the V3 region of the 16...

AI Technical Summary

Problems solved by technology

However, the method of sequencing by transforming and cloning vectors has cumbersome test steps, long test period, heavy workload, and high cost. The most serious disadvantage is that there may be errors in obtaining sequence information.

There are probably two reasons for the error: first, low-abundance pollution occurs during the secondary amplification of the DNA template, and the contaminated sequence is mistakenly taken as the target sequence due to improper selection of positive clones; on the other hand, the DNA polymerase itself uses DNA as a template. Missynthesis can also occur during catalyzed semi-conservative replication, and the accuracy of the sequence may be reduced when selecting positive single clones for sequencing

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