摘要
目的建立食品中椰毒假单胞菌酵米面亚种实时荧光PCR检测方法。方法根据椰毒假单胞菌酵米面亚种16S^23S rRNA基因片段序列,用Oligo7设计一对特异性引物和一个TaqMan探针,摸索最佳退火温度,最佳引物和探针浓度,建立椰毒假单胞菌酵米面亚种实时荧光PCR检测方法。通过对唐菖蒲伯克霍尔德氏菌以及22种其他标准菌株进行实时荧光PCR检测,验证本法的特异性和抗干扰性。同时从菌液浓度和DNA浓度水平上进行研究,确定该检测方法的灵敏度。结果用该方法检测23种菌,除唐菖蒲伯克霍尔德氏菌外,其他22种细菌均为阴性。抗干扰性试验显示杂菌对检测结果不影响,本法抗干扰能力强。通过灵敏度试验的研究,确定本法检测椰毒假单胞菌酵米面亚种的菌液灵敏度为1×10^2 CFU/mL,DNA灵敏度为250 fg/μL。结论本试验设计的引物和探针具有较强特异性、抗干扰性以及灵敏度,该方法具有很好的研究价值和应用前景。
Objective To develop a fluorescence real-time PCR method to detect Pseudomonas cocovenenans subsp.Farinofermentans in food.Methods Species-specific primers and TaqMan fluorescent probe were designed by oligo7 software according to the conserved sequence of 16S^23S rRNA of Pseudomonas cocovenenans subsp.farinofermentans.The optimum annealing temperature,probe and primer concentration were explored.A real-time fluorescent PCR detection method for Pseudomonas cocovenenans subsp.farinofermentans was established.The specificity and anti-interference of this method were verified by real-time PCR detection of Burkholderia gladiolus and 22 other standard strains.At the same time,the sensitivity of the method was determined by studying the concentration of bacterial solution and DNA.Results This method was used to detect 23 kinds of bacteria,it showed that except Burkholderia gladiolus,22 kinds of bacteria were negative.The anti-interference test showed that the mixed bacteria had no effect on the detection result,and the method had strong anti-interference ability.The sensitivity of fluorescence real-time PCR to detect Pseudomonas cocovenenans subsp farinofermentans was 1×10^2 CFU/mL and that of DNA was 250 fg/μL.Conclusion The primers and probe designed in this study have strong specificity,anti-interference quality and sensitivity.This method is valuable for research and application prospects.
作者
林捷
方陈玉
陆晶芳
尹丹韩
赵奇琦
王加胜
LIN Jie;FANG Chen-Yu;LU Jing-Fang;YIN Dan-Han;ZHAO Qi-Qi;WANG Jia-Sheng(Hangzhou Institute for Food and Drug Control,Hangzhou 310000,China)
出处
《食品安全质量检测学报》
CAS
2020年第11期3538-3544,共7页
Journal of Food Safety and Quality
