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

  • YANG Xiangqian ,
  • CUI Jingchun ,
  • ZENG Shixian ,
  • CHENG Li ,
  • ZHANG Kebin ,
  • HU Xiaohong ,
  • BAO Yajing
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  • 1. College of Life Science, Dalian Minzu University, Dalian 116600, China;
    2. College of Environment and Resources Sciences, Dalian Minzu University, Dalian 116600, China

Received date: 2020-06-13

  Online published: 2021-01-18

Abstract

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 9.33×105 copies/g on the first day of fermentation and 4.16×108 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.

Cite this article

YANG Xiangqian , CUI Jingchun , ZENG Shixian , CHENG Li , ZHANG Kebin , HU Xiaohong , BAO Yajing . Development of Real-Time Fluorescent Quantitative PCR Detection Method for Bacillus amyloliquefaciens and Its Application in Fermented Soybean Meal[J]. Chinese Journal of Animal Nutrition, 2021 , 33(1) : 494 -505 . DOI: 10.3969/j.issn.1006-267x.2021.01.050

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