牛源微生物样本灭活保存液的制备及在荧光定量PCR检测中的适用性评估

Preparation of inactivation preservation solution for bovine-derived microbial samples and its application evaluation in real-time fluorescent quantitative PCR

摘要

在动物疫病诊断与监测中，临床样本的规范采集与处理是保障病原准确检测与鉴定的关键环节。为了解决拭子类病料在运输过程中的保存难题，本研究研制了一种以胍盐为主的微生物样本灭活保存液，适用于实时荧光定量PCR（qPCR）检测的前处理。选取金黄色葡萄球菌、无乳链球菌、多杀性巴氏杆菌和牛支原体作为代表性病原，分别在4℃、22℃和37℃条件下，评估该微生物样本灭活保存液在1、3和7d内的保存效果。结果显示：该微生物样本灭活保存液可迅速灭活上述病原，并在常温（22℃）下至少保存7d仍能有效抑制核酸降解；经4℃保存3d后，多种病原的qPCR检测灵敏度仍可维持在10 ²～10 ⁴ CFU/mL。综上，本研究所制备的微生物样本灭活保存液具备快速灭活与稳定保护核酸的双重功能，可有效降低运输过程中的生物安全风险，保障下游分子检测的可靠性，适用于临床样本的现场采集与实验室检测。

Abstract

In the diagnosis and monitoring of animal diseases, the standardized collection and processing of clinical samples are crucial links to ensuring accurate detection and identification of pathogens. To address the preservation challenge of swab-based disease samples during transportation, this study developed a guanidine salt-based microbial sample inactivation and preservation solution, which is helpful for pretreatment of real-time fluorescent quantitative PCR (qPCR) detection. Staphylococcus aureus, Streptococcus agalactiae, Pasteurella multocida, and Mycoplasma bovis were selected as representative pathogens. The preservation effect of this solution was evaluated at 4℃, 22℃, and 37℃ for 1 day, 3 days, and 7 days, respectively. The results showed that this preservation solution was able to quickly inactivate the above-mentioned pathogens and to effectively inhibit nucleic acid degradation for at least 7 days at room temperature (22℃). After being stored at 4℃ for 3 days, the qPCR detection sensitivity of various pathogens could still be maintained at 10 ²-10 ⁴ CFU/mL. The inactivation and preservation solution prepared in this study possessed dual functions of rapid inactivation and stable nucleic acid protection. It would effectively reduce the biosafety risk during transportation, ensure the reliability of downstream molecular detection, and would be applicable for on-site collection of clinical samples and laboratory detection.

关键词

微生物样本灭活保存液 / qPCR / 制备 / 病原灭活

Key words

microbial preservative / qPCR / preparation / pathogen inactivation

参考文献

[1] THRUFSIELD M, CHRISTLEY R. Veterinary epidemiology [M]. Fourth Edition, John Wiley & Sons Ltd., 2018.

[2] ROCHELLE P A, CRAGG B A, FRY J C, et al. Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis [J]. FEMS Microbiol Ecol, 1994, 15(1/2): 215-225.

[3] SEKAR R, KACZMARSKY L T, RICHARDSON L L. Effect of freezing on PCR amplification of 16S rRNA genes from microbes associated with black band disease of corals [J]. Appl Environ Microbiol, 2009, 75(8): 2581-2584.

[4] LIN A B, FLETCHER M A, VALETTE M, et al. A Triton X-100-split virion influenza vaccine is safe and fulfills the committee for proprietary medicinal products (CPMP) recommendations for the European community for immunogenicity, in children, adults and the elderly [J]. Biologicals, 2000, 28(2): 95-103.

[5] LEIGH W J, ZADOKS R N, COSTA J Z, et al. Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene [J]. J Appl Microbiol, 2020, 129(1): 63-74.

[6] THOMAS A C, BAILEY M, LEE M R F, et al. Insights into Pasteurellaceae carriage dynamics in the nasal passages of healthy beef calves [J]. Sci Rep, 2019, 9(1): 11943.

[7] 钟辉, 姚火春. 奶牛乳腺炎金黄色葡萄球菌临床分离株生化特性与菌体蛋白免疫原性分析 [J]. 农业生物技术学报, 2006, 14(6): 970-975.

[8] 马彦颖, 高磊, 宫枫举, 等. 牛支原体、多杀性巴氏杆菌、溶血性曼氏杆菌三重qPCR方法的建立与流行病学调查 [J]. 微生物学报, 2025, 65(9): 4198-4210.

[9] 王斌, 李秀艳. 一种灭活型病毒保存液配方及其制作方法: CN113186251A [P]. 2021-07-30.

[10] 陈裕殷, 高芸, 夏广亮, 等. 用于基因检测的微生物样本保存液及其制备方法、试剂盒与应用: CN108546646B [P]. 2021-08-31.

[11] PRADIER E. An easy method for preserving nucleic acids in field samples for later molecular and genetic studies without refrigerating [J]. J Evol Biol, 1995, 8(5): 657-663.

[12] BICOCCA V T, LEVIN T G. Methods and compositions for collection and preservation of biologicals: US20220389405A1 [P]. 2022-12-15.

[13] RHEE S S, BURKE D H. Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry [J]. Anal Biochem, 2004, 325(1): 137-143.

基金资助

甘肃省科技计划技术创新引导计划项目(25CXNJ009)

山东省牛产业体系项目(SDAIT-09-07)