牛奶中布鲁氏菌和单增李斯特菌双重PCR检测方法的建立  

Establishment of multiplex PCR for detecting Brucella and Listeria monocytogenes in raw milk

作　　者：程媛媛[1] 张彦明[1] 向华[1] 康恺[1] 李艳明[1] 徐磊[1] 董玲娟[1] 张三东[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2011年第3期54-60,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家公益性行业(农业)科研专项(200903055)

摘　　要：【目的】建立牛奶中布鲁氏菌和单增李斯特菌的双重PCR检测方法。【方法】根据布鲁氏菌IS711序列和单增李斯特菌毒力因子HlyA序列设计并合成引物,建立双重PCR检测方法,对其特异性、敏感性和重复性进行检测,并制备污染模型乳,通过直接检测、增菌后检测、细菌富集后增菌检测,确定最低检测限。【结果】建立了牛奶中布鲁氏菌和单增李斯特菌双重PCR检测方法,该方法特异性好、敏感性高、可重复性好。布鲁氏菌和单增李斯特菌单PCR检测最小DNA质量浓度分别为0.282和3.33 pg/μL,双重PCR检测最小DNA质量浓度分别为2.82和33.3 pg/μL。感染模型乳不经过增菌,布鲁氏菌和单增李斯特菌最低检测限分别为104和8×104CFU/mL;经过24h增菌,就可以检测到10 CFU/mL的布鲁氏菌,经过12 h增菌,就可以检测到8 CFU/mL的单增李斯特菌;经过离心法富集细菌后再增菌,灵敏度可以扩大40~50倍,布鲁氏菌经24 h增菌后最低检测限为10 CFU/40 mL,单增李斯特菌经12 h增菌后最低检测限为8 CFU/40 mL。【结论】建立的双重PCR检测方法特异、灵敏、可重复,可单独或同时检测牛奶中的布鲁氏菌和单增李斯特菌,也可以用于其他产品中布鲁氏菌和单增李斯特菌的检测。【Objective】 The research was done to establish a multiplex PCR method for detecting Brucella and Listeria monocytogenes in raw milk.【Method】 Two pairs of primers were designed according to IS711 gene of Brucella,HlyA gene of L.monocytogenes,then the multiplex PCR method was established and performed to detect its specificity,sensibility and repeatability.Contamination model was also prepared to determine the lowest detecable limit of Brucella and L.monocytogenes by bacteria culturing and gathering.【Result】 The multiplex PCR method was established and it was specific,sensitive and well-repeatable.This PCR assay could detect as low as 0.282 pg/μL DNA of Brucella and 3.33 pg/μL DNA of L.monocytogenes in independent PCR and 2.82 pg/μL DNA of Brucella and 33.3 pg/μL DNA of L.monocytogenes in multiplex PCR.Further study showed if the bacteria were not concentrated and cultured,this method of assay could detect as few as 104 CFU/mL of Brucella and 8×104 CFU/mL of L.monocytogenes in contaminated milk.When the contaminated milk was cultured for 24 hours,this method could detect as few as 10 CFU/mL of Brucella and 8 CFU/mL of L.monocytogenes when it was cultured for 12 hours.If bacteria were concentrated and gathered from raw milk by centrifuge,the sensitivity could amplify 40-50 times.When the contaminated milk was cultured for 24 hours after gathering,this method could detect as few as 10 CFU/40 mL of Brucella,if it was cultured for 12 hours after gathering,this method could detect as few as 8 CFU/40 mL of L.monocytogenes.【Conclusion】 This multiplex PCR method was specific,sensitive and well-repeatable to detect Brucella and L.monocytogenes in raw milk or in other things respectively or simultaneously.

关 键 词：布鲁氏菌 单增李斯特菌 双重PCR 牛奶 

分 类 号：S852.614[农业科学—基础兽医学]