Effects of Different Sampling Methods of Chilled Chickens on the Microbial DNA Extraction and the High-throughput Sequencing Results

Abstract

Abstract: Objective: To compare the swabbing and rinsing methods used for sampling of chilled chickens by evaluating their effects on the microbial DNA extraction and the subsequent high-throughput sequencing. Methods: Four chilled chickens were divided into equal halves and sampled by superficial swabbing and rinsing methods respectively for each half. Microbial genomic DNA was extracted from the collected samples and the V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR. The PCR products were then subjected to high-throughput sequencing on an Illumina HiSeq sequencing platform．The obtained sequences were processed and analyzed using QIIME and other software. Results: There was no difference in bacterial genomic DNA extraction and bacterial community richness, diversity and structures between the samples collected by the two methods (P＞0.05). High-throughput sequencing showed that the bacterial community in chilled chicken samples consisted mainly of 7 phyla and 10 main genera. Proteobacteria was the dominant phylum, accounting for more than 70% of the bacterial community; Shewanella, Pseudomonas, Acinetobacter, Psychrobacter, and Brochothrix were the dominant genera, each representing 10% ~ 20% of the bacterial community. Conclusions: The two sampling methods of chilled chicken exhibit no obvious difference in the bacterial DNA extraction and high-throughput sequencing results. However, compared with swabbing, rinsing method has the advantages of easily operating and non-secondary-contamination, so it is more practical in microbiological analysis of chilled chicken. The experiment also provides data for the study of surface bacteria of chilled chicken.

Key words: chilled chicken, bacterial community, swabbing method, rinsing method, high-throughput sequencing

CLC Number:

Xiang-Xiang HE. Effects of Different Sampling Methods of Chilled Chickens on the Microbial DNA Extraction and the High-throughput Sequencing Results[J]. FOOD SCIENCE, 0, (): 0-0.

References

[1] 吴海虹, 刘朏, 刘芳, 等. 低温贮藏对冷鲜鸡腐败菌菌群变化的影响[J]. 现代食品科技, 2016(4): 177-181.
[2] 易鹤. 现宰鸡、冷冻鸡和冷鲜鸡[J]. 农村新技术, 2014(6): 57.
[3] SEGATA N, BOERNIGEN D, TICKLE T L, et al. Computational meta'omics for microbial community studies[J]. Molecular Systems Biology, 2013, 9: 666.
[4] 张赫宇, 杨波, 罗瑞明, 等. 高通量测序分析冷鲜滩羊肉储藏过程中的细菌群落多样性[J].食品工业科技, 2016, 37(13): 177-182.
[5] CAPORASO J G, KUCZYNSKI J, STOMBAUGH J, et al. QIIME allows analysis of high-throughput community sequencing data[J]. Nature methods, 2010, 7(5): 335-336.
[6] BOKULICH N A, SUBRAMANIAN S, FAITH J J, et al. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing[J]. Nature Methods, 2013, 10(1): 57-59.
[7] EDGAR R C. UPARSE: highly accurate OTU sequences from microbial amplicon reads[J]. Nature methods, 2013, 10(10): 996-998.
[8] COSSI M V, DE ALMEIDA M V, DIAS M R, et al. Comparison of destructive and nondestructive sampling techniques of retail chicken carcasses for enumeration of hygiene indicator microorganisms[J]. Journal of Food Protection, 2012, 75(1): 29-33.
[9] ZHANG Q Q, YE K P, XU X L, et al. Comparison of excision, swabbing and rinsing sampling methods to determine the microbiological quality of broiler carcass[J]. Journal of Food Safety, 2012, 32(1):134-139.
[10] GIOMBELLI A, CAVANI R, GLORIA M B. Evaluation of three sampling methods for the microbiological analysis of broiler carcasses after immersion chilling[J]. Journal of Food Protection, 2013, 76(8): 1330-1335.
[11] 商宝娣, 杨星, 李正友, 等. 希瓦氏菌的研究进展[J]. 福建农业, 2015, (7): 152-154.
[12] 高磊, 谢晶, 叶藻, 等. 冷鲜鸡腿肉中优势腐败菌的分离鉴定及腐败能力研究[J]. 食品与发酵工业, 2015, 41(8): 48-53.
[13] OLOFSSON T C, AHRNE S, MOLIN G. Composition of the bacterial population of refrigerated beef，identified with direct 16S rRNA gene analysis and pure culture technique[J]. International Journal of Food Microbiology, 2007, 118(3): 233-240.
[14] 曾晓房, 林惠珍, 邝智祥, 等.冷鲜肉中腐败菌的研究现状[J].安徽农业科学, 2010, 38(34):19550 -19552.
[15] 江芸, 高峰, 徐幸莲, 等. 冷却猪肉不同前处理对细菌DNA提取及PCR-DGGE的影响[J]. 食品科学, 2010, 31(6): 258-262.
[16] 梁荣蓉. 生鲜鸡肉调理制品菌群结构分析和产品货架期预测模型的研究[D]. 泰安: 山东农业大学, 2010.
[17] 蓝蔚青, 谢晶, 周会, 等. 不同时期鲳鱼冷藏期间优势腐败菌的多样性变化[J]. 食品科学, 2015(2): 226-231.
[18] 倪萍, 纪文营, 黄洁洁, 等. 冷却猪肉中热死环丝菌的分离、鉴定及致腐能力[J]. 食品科学, 34(21): 140-144.
[19] PENNACCHIA C, ERCIKUBU D, VILLANI F. Development of a Real-Time PCR assay for the specific detection of Brochothrix thermosphacta in fresh and spoiled raw meat[J]. International Journal Food Microbiology, 2009, 134 (3): 230-236.