| Microorganisms contamination occurs when cultured in the farm and under the procedure of harvesting,processing,transportation and storage.Microorganisms affects the quality and stability of Chinese herbal medicines(CHM).For the quality control of microorganisms in CHM,the commonly used method is to test in accordance with the general principles of the Chinese pharmacopoeia(2015),"1105 microbial limit test for non-sterile products:microbial count method" and "1106 microbial limit test for non-sterile products:control bacteria test" and "1107 microbial limit standard for non-sterile drugs".The traditional culture-dependent methods suffers from time consuming,poor accuracy,low efficiency,labor intensive,and most of the microorganisms cannot be cultured under laboratory conditions due to their own characteristics.At the present time,the microbial limit standard specifically requires that Salmonella(10 g)should not be detected in the powder by oral administration of expensive fine CHM,direct oral administration and soaked CHM,and that the bile-resistant gram-negative bacteria should be less than 104 cfu(1 g),while methods for other microorganisms still to be developed.Information of the microbial community is of great value for assessing the risk of microbial contamination and establishing the quality control standards of CHM.Therefore,it is essential to study on the rapid detection technologies for profiling microorganisms in CHM.In this paper,high-throughput gene detection technologies is applied to the detection of microorganism species in CHM.Those methods profiling the microorganisms in CHM by a culture-independent method-gene detection on microbial DNA,which greatly shortens the detection time,and expanded the detection range.Firstly,high-throughput gene microarray method was used to investigate the contamination of microorganisms and analyze the microbial species composition in 11 kinds of CHM.A total of 171 species were detected,which consisted of bacteria,fungi,and virus.Bacteria were the most dominant fraction,which accounted for 75.44%of total microorganism.The most abundant generals were Enterobacter,Pseudomonas and Bacillus in all bacteria generals.Secondly,according to the detection results of high-throughput gene microarray method,the method of bacteria 16S rDNA amplicon library construction was established based on the existing Ion S5 sequencing platform in the laboratory and applied to detect bacterial species in CHM.The ratio of effective tags was used to evaluate different library construction methods.Results indicated that the quality of library was slightly affected by pcr circle,beads from different supplier and amount of starter template DNA.However,DNA polymerase had a significant impact The analysis of the sequencing data of known Mock DNA showed that the vast majority of species in the 16 bacterial communities could be detected,indicated that the method was applicable for bacterial detection and diversity analysis in the samples.Bacteria 16S rDNA amplicon high-throughput sequencing method of Ion S5 sequecing platform was applied to analyze the bacterial species in CHM,the results showed that there were about 200 species of bacteria in 11 kinds of CHM,and the most abundant generals were Enterobacter,Pseudomonas and Acinetobacter.Finally,we compared the results of bacterial species in CHM by the two methods.The two methods shows consistency in the detection of main genera.The bacterial 16S rDNA amplicon high-throughput sequencing method detected more taxa,while the high-throughput gene microarray method was more sensitive though it might suffer from false positive in the dectection of unknown microorganisms.The two methods were applicable to detect microorganisms in CHM,The high-throughput gene microarray method preferred known bacteria,fungi,and virus detection with well designed probes,while 16S rDNA amplicon high-throughput sequencing method is fit for bacteria community profiling without the limitations of predesign. |
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