Randomly Amplified Polymorphic DNA (RAPD) Analysis of Lactic Acid Bacteria from Yak Milk Cheese

doi:10.7506/spkx1002-6630-201307043

Abstract

Abstract: Objective: To explore the application value of randomly amplified polymorphic DNA (RAPD)-PCR for the genotype evaluation of lactic acid bacteria isolated from yak milk cheeses. Methods: In order to establish a reliable RAPD protocol with primer M13, factors such as annealing temperature, Mg2+ concentration, primer amount, Taq polymerase and DNA template for affecting the quality and reproducibility of RAPD profiles were investigated. The optimal PCR procedure and reaction reagent concentration were employed for RAPD amplification of 39 isolates from yak milk cheese on the basis of previous identification of 16S rRNA sequence analysis. The electrophoretic profiles were analyzed using NTsys2.10e software, genetic similarity coefficients were calculated and a cluster dendrogram was plotted. Results: Genetic similarity coefficients of 39 strains ranged from 0.47 to 0.98. The cluster dendrogram showed that 39 isolates were classified into 10 groups using the similarity coefficient of 0.78 as a cut-off point. The discrimination of groups was consistent with the assignment of the species based on 16S rRNA sequence analysis, except for strains X23, Y36 and Y37. Conclusion: RAPD technique is useful in taxonomy of lactic acid bacteria isolated from yak milk cheese. The accuracy of species identification can be improved by the combination of RAPD with other kinds of genotypic methods.

Key words: yak milk cheese；lactic acid bacteria；randomly amplified polymorphic DNA (RAPD)；cluster analysis, genotype；genetic similarity coefficient

CLC Number:

TS252.54

YANG Ji-xia1，CHEN Zhi-lan2，YANG Hai-yan3，HUANG Ai-xiang4，CHEN Zong-dao1. Randomly Amplified Polymorphic DNA (RAPD) Analysis of Lactic Acid Bacteria from Yak Milk Cheese[J]. FOOD SCIENCE, 2013, 34(7): 206-211.

References

[1]WILLIAMS J G K, KUBELIK A R, LIVAK K J, et at.DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J].Nucleic Acids Research,1990,18(22):6531-6535
[2]WELSH J, MCCLELLAND M.Fingerprinting genomes using PCR with arbitrary primers[J].Nucleic Acids Research,1990,18(24):7213-7218
[3]NIGATU A, AHRNé S, MOLIN G.Randomly amplified polymorphic DNA (RAPD) profiles for the distinction of Lactobacillus species[J].Antonie van Leeuwenhoek,2001,79:1-6
[4]PROD?LALOVá J, ?PANOVá A, RITTICH B.Application of PCR, rep-PCR and RAPD techniques for typing of Lactococcus lactis strains[J].Folia Microbio,2005,50(2):150-154
[5]MARTIN B, COROMINAS L, GARRIGA M, et al.Identification and tracing of Enterococcus sppby RAPD-PCR in traditional fermented sausages and meat environment[J].. Journal of Applied Microbiology,2009,106:66-77
[6]CIBIK R, LEPAGE E, TAILLIEZ P.Molecular diversity of Leuconostoc mesenteroides and Leuconostoc citreum isolated from traditional French Cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification[J].Systematic and Applied Microbiology,2000,23(2):267-278
[7]马成杰，吴正钧，杜昭平，等.不同品牌酸乳中德氏乳杆菌的分离鉴定及RAPD分析[J].食品科学,2010,31(7):177-181
[8]TYLER KD, WANG G, TYLER SD, et al.Factors affecting reliability and reproducibility of amplification- based DNA fingerprinting of representative bacterial pathogens[J].Journal of Clinical Microbiology,1997,35(2):339-346
[9]MICHELI MR, BOVA R, PASCALE E, et al.Reproducible DNA fingerprinting with the random amplified polymorphic DNA (RAPD) method[J].Nucleic Acid Research,1994,22(10):-
[10]李平兰.[J].贺稚非. 食品微生物学实验原理与技术[M]. 北京: 中国农业出版社,2005,:-
[11]PITCHER DG, SAUNDERS NA, OWEN RJ.Rapid extraction of bacterial genomic DNA with guanidium thiocyanate[J].Letters in Applied Microbiology,1989,8:151-156
[12] 林万明. 细菌分子遗传学分类鉴定法[M]. 上海: 上海科学技术出版社, 1990: 48-83
[13]朱红梅，廖万清，戴建新，等.用微小卫星引物PCR鉴定红色毛癣菌和须癣毛癣菌[J].第二军医大学学报,2001,22(3):252-254
[14]STILES ME, HOLZAPFEL WH.Lactic acid bacteria of foods and their current taxonomy[J].International Journal of Food Microbiology,1997,36(1):1-29
[15]COCCONCELLI PS, PORRO D, GALANDINI S, et al.Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci[J].Letters in Applied Microbiology,1995,21(6):376-379
[16]PLESSIS EM, DICKS LM.Evaluation of Random Amplified Polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and lactobacillus johnsonii[J].Current Microbiology,1995,31(2):114-118
[17]JOHANSSON ML, QUEDNAU M, MOLIN G, et al.Randomly amplified polymorphic DNA (RAPD) for rapid typing of lactobacillus plantarum strains[J].Letters in Applied Microbiology,1995,21(3):155-159
[18]VAN REENEN CA, DICKS LM.Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus plantarum and Lactobacillus pentosus[J].Current Microbiology,1996,32(4):183-187
[19]WARD LJ, TIMMINS MJ.Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction[J].Letters in Applied Microbiology,1999,29(2):90-92