解淀粉芽孢杆菌实时荧光定量PCR测定方法的建立及其在豆粕发酵中的应用

本试验旨在通过快速定量不同发酵时期下发酵豆粕中解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的数量,以解决豆粕发酵过程品质监控的瓶颈难题,建立解淀粉芽孢杆菌的实时荧光定量PCR方法。根据解淀粉芽孢杆菌DNA解旋酶A亚基(gyrA)基因及16S核糖体RNA(16S rRNA)基因的保守区设计出3对引物,利用常规PCR筛选出1对特异性引物,并以该引物的扩增产物构建的重组质粒作为标准品,建立实时荧光定量PCR方法,并对该方法进行特异性、灵敏性、重复性检验,核酸水平抗干扰验证及豆粕对检测敏感度的干扰验证,最后对发酵的豆粕样品进行定量检测。结果表明:1)以gyrA基因设计的引物构建的质粒标准品建立的实时荧光定量PCR方法具有良好的特异性;2)该方法检测解淀粉芽孢杆菌核酸和菌液的最低检测限分别为102 copies/μL和103 CFU/mL;3)该方法组内变异系数在0.76%~3.27%,组间变异系数在0.34%~1.88%,均低于5%,说明重复性良好;4)含有与不含副干酪乳杆菌核酸的解淀粉芽孢杆菌的Ct值无显著差异(P>0.05),说明该方法不受检样中其他微生物的核酸干扰;5)该方法对豆粕中解淀粉芽孢杆菌的检测限为104 CFU/mL,比纯菌液最低检测敏感度(103 CFU/mL)降低了1个数量级(但不影响该方法的实际检测应用)。解淀粉芽孢杆菌在不同发酵时期发酵豆粕样品中定量检测结果表明,发酵0 d解淀粉芽孢杆菌的数量为9.33×10^5 copies/g,发酵至5 d接种菌数量为4.16×10^8 copies/g。由此可见,本试验建立的解淀粉芽孢杆菌实时荧光定量PCR方法特异性强,灵敏度高,抗干扰能力强,可快速定量发酵豆粕中的解淀粉芽孢杆菌的数量。

Development of Real-Time Fluorescent Quantitative PCR Detection Method for Bacillus amyloliquefaciens and Its Application in Fermented Soybean Meal

A new real-time fluorescent quantitative PCR(qPCR)detection method for detection of the quantity of Bacillus amyloliquefaciens in fermented soybean meal at different fermentation stages was developed in our study to solve the bottleneck problem of quality monitoring in the fermentation process of soybean meal.Three pairs of primers were designed from the conservative region of gyrase subunit A(gryA)gene and 16S ribosomal RNA gene of B.amyloliquefaciens.A pair of specific primers were screened out by conventional PCR,and the recombinant plasmid constructed by the amplification products of the primers was used as the standard.Then a real-time fluorescent quantitative PCR method was established.The established method was assessed with respect to specificity,sensitivity and repeatability,and the level of nucleic acid anti-interference,interference of soybean meal detection sensitivity.Finally,it was applied to detection of the fermented soybean meal samples.The results showed as follows:1)the real-time fluorescent quantitative PCR method based on plasmid standard constructed with primers designed by gyrA gene had good specificity;2)the minimum detection limits of nucleic acid level and bacterial solution of B.amylolyquefaciens were 102 copies/μL and 103 CFU/mL,respectively;3)the coefficient variation(CV)were 0.76%to 3.27%in the intragroup and 0.34%to 1.88%in the intergroup,all of which were lower than 5%,indicating good repeatability;4)there was no significant difference in Ct value between B.amylolyquefaciens with and without nucleic acid of Lactobacillus paracasei(P>0.05),indicating that the method was not interfered by nucleic acid of other microorganisms in the sample;5)the detection limit of this method for B.amylolyquefaciens in soybean meal was 104 CFU/mL,which was one order of magnitude lower than the lowest detection sensitivity(103 CFU/mL)of pure bacterial solution(but did not affect the actual detection application of this method).The results showed that the number of B.amylolyquefaciens was 9.33×10^5 copies/g on the first day of fermentation and 4.16×10^8 copies/g on the fifth day of fermentation.Comprehensively,the results indicate that the established qPCR method has good specificity,sensitivity,anti-interference ability,and it can rapidly quantify the B.amyloliquefaciens in fermented soybean meals.