贵州省畜禽源葡萄球菌和链球菌快速鉴定双重PCR方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2019.03.028

Abstract

Abstract:

In order to establish a duplex PCR method for accurately and quickly identifying common pathogens Staphylococcus and Streptococcus in clinical cases of livestock and poultry,this experiment selected nuc gene of Staphylococcus and EF-TU gene conserved fragments of Streptococcus to design a pair of specific primers,construct a duplex PCR system which could simultaneously identify Staphylococcus and Streptococcus,optimize the reaction conditions,screen out the optimal primer concentration and annealing temperature;Using this method to detect 73 other gram-positive clinical isolates,the specificity of the method was evaluated;The sensitivity of the detection method was determined by diluting and counting the cultured Staphylococcus and Streptococcus;The detection method was used to detect the clinical isolates of Staphylococcus and Streptococcus of livestock and poultry in Guizhou province.The results showed that the optimal primer addition amount was 1 μL,and the optimal annealing temperature was 56 ℃.The other 73 strains showed no amplification bands,and the duplex PCR method was better.The specificity of the sensitivity test showed that the sensitivity of Staphylococcus and Streptococcus were 1.50 ng/μL and 1.44 pg/μL,respectively.The retest results of clinical samples showed that there were 40 strains of Staphylococcus (54.79%) and 33 strains of Streptococcus (45.21%) in 73 clinical isolates,the coincidence rate with traditional bacteria separation and identification method was 97.26%.This experiment established a duplex PCR method for specific,sensitive and rapid identification of Staphylococcus and Streptococcus pathogens in Guizhou province,providing effective techniques for rapid diagnosis and epidemiological investigation of clinical cases.

Key words: Staphylococcus; Streptococcus; duplex PCR

CLC Number:

S852.61⁺1

ZHANG Yundan, MA Guangqiang, YUE Jun, ZHOU Bijun, WEN Ming, CHENG Zhentao. Establishment and Application of Duplex PCR for Rapid Identification of Staphylococcus and Streptococcus of Livestock and Poultry in Guizhou Province[J]. , 2019, 46(3): 873-880.

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Establishment and Application of Duplex PCR for Rapid Identification of Staphylococcus and Streptococcus of Livestock and Poultry in Guizhou Province

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