[1]赵伟凯,王子怡,刘文静,等.呼吸道样本NaOH液化处理对核酸检测的影响及方法优化选择[J].现代检验医学杂志,2026,41(01):175-179.[doi:10.3969/j.issn.1671-7414.2026.01.034]
　ZHAO Weikai,WANG Ziyi,LIU Wenjing,et al.Impact of NaOH Liquefaction Treatment on Nucleic Acid Testing of Respiratory Samples and the Optimization of Method Selection[J].Journal of Modern Laboratory Medicine,2026,41(01):175-179.[doi:10.3969/j.issn.1671-7414.2026.01.034]
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呼吸道样本NaOH液化处理对核酸检测的影响及方法优化选择()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第41卷
期数:
2026年01期
页码:
175-179
栏目:
论著
出版日期:
2026-01-15

文章信息/Info

Title:
Impact of NaOH Liquefaction Treatment on Nucleic Acid Testing of Respiratory Samples and the Optimization of Method Selection
文章编号:
1671-7414（2026）01-175-05
作者:
赵伟凯1,2，王子怡1，刘文静1，孙玉杰1，刘仪威1，徐英春1，陈 雨1，杨启文1，伊 洁1
1.中国医学科学院北京协和医院检验科，北京100730;2.北京怀柔医院检验科，北京101400
Author(s):
ZHAO Weikai1,2，WANG Ziyi1，LIU Wenjing1，SUN Yujie1，LIU Yiwei1，XU Yingchun1，CHEN Yu1，YANG Qiwen1，YI Jie1
1.Department of Laboratory Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China;2.Department of Laboratory Medicine, Beijing Huairou Hospital, Beijing 101400, China
关键词:
氢氧化钠; 液化; 核酸; 聚合酶链反应; 三羟甲基氨基甲烷盐酸盐
分类号:
R730.53；R979.1
DOI:
10.3969/j.issn.1671-7414.2026.01.034
文献标志码:
A
摘要:
目的探讨呼吸道样本NaOH处理对核酸检测效率的影响及优化方法选择，为呼吸道样本核酸提取前处理流程提供科学依据。方法从北京协和医院检验科收集16份鼻咽拭子（NPS）样本和24份痰液样本。将浓度为1×106copies/ml的肺炎支原体（MP）DNA国家标准品分别用病毒保存液和痰液稀释，制备成模拟阳性鼻咽拭子样本和模拟阳性痰液样本。临床鼻咽拭子样本和模拟鼻咽拭子样本分别按照1∶1比例使用生理盐水、1mol/LNaOH，胰酶进行液化处理。模拟痰液样本按照1∶1比例分别用胰酶和1mol/LNaOH液化处理。模拟痰液样本用1mol/LNaOH液化后，加不同浓度三羟甲基氨基甲烷盐酸盐[Tris（hydroxymethyl）aminomethanehydrochloride，Tris-HCl]优化处理。所有样本均使用普通磁珠法核酸提取和聚合酶链式反应（polymerasechainreaction，PCR）扩增。运用t检验比较不同液化试剂处理样本后核酸Ct值的组间差异。结果与生理盐水相比，1mol/LNaOH处理显著抑制鼻咽拭子中甲型流感病毒（IFVA）和呼吸道合胞病毒（RSV）RNA的检测,结果由阳性转为阴性；而处理前后MP和腺病毒（ADV）DNA的Ct值差异无统计学意义（t=2.644、2.862，均P>0.05）。在模拟鼻咽拭子样本中，1mol/LNaOH液化处理对2000和1000copies/mlMPDNA无影响（t=0.946、1.925，均P>0.05），但对低载量（200和500copies/ml）的检测具有显著抑制作用（t=3.085、2.566，均P<0.05）。胰酶处理对DNA和RNA病原体核酸检测均无显著影响（均P>0.05）。模拟痰液样本经1mol/LNaOH液化后，MPDNACt值相比胰酶组显著升高（t=3.935，P<0.05），使用不同浓度（1、0.5和0.25mol/L）Tris-HCl（pH=7）中和后Ct值差异无统计学意义（t=0.333、1.984、0.182，均P>0.05）。结论1mol/LNaOH液化痰液对PCR检测具有抑制作用，可能导致假阴性结果。通过添加Tris-HCl可以一定程度缓冲NaOH的抑制，提高PCR检测的准确性。
Abstract:
Objective To investigate the impact of NaOH treatment on respiratory samples for nucleic acid testing efficiency and the selection of optimization methods, providing a scientific basis for the pre-treatment process of nucleic acid extraction from respiratory samples. Methods 16 nasopharyngeal swabs(NPS) and 24 sputum samples were collected from the Department of Clinical Laboratory of Peking Union Medical College Hospital. The national standard of Mycoplasma pneumoniae (MP) DNA with a concentration of 1×106 copies/ml was diluted with viral transport medium and sputum respectively to prepare simulated positive NPS samples and simulated positive sputum samples. Clinical NPS samples and simulated NPS samples were liquefied with normal saline(NS), 1mol/L NaOH, and trypsin at a 1∶1 ratio respectively. The simulated sputum samples were liquefied with trypsin and 1 mol/L NaOH at a 1∶1 ratio, respectively. After liquefaction with 1 mol/L NaOH, the simulated sputum samples were treated with varying concentrations of Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) for optimization. All samples underwent nucleic acid extraction using the conventional magnetic bead method followed by polymerase chain reaction (PCR) amplification. A t-test was employed to compare the intergroup differences in nucleic acid Ct values among samples treated with different liquefaction reagents. Results Compared with NS, treatment with 1 mol/L NaOH significantly inhibited the detection of influenza A virus（IFVA）and respiratory syncytial virus（RSV）RNA, converting the results from positive to negative, whereas the differences in Ct values of MP and adenovirus (ADV) DNA before and after treatment showed no statistical significance (t=2.644, 2.862, all P>0.05). In simulated NPS samples, 1 mol/L NaOH liquefaction treatment showed no effect on MP DNA at 2 000 and 1 000 copies/ml (P>0.05), but exhibited significant inhibitory effects on detection at low viral loads (200 and 500 copies/ml) (t=3.085、2.566, all P<0.05). Trypsin treatment had no significant impact on nucleic acid detection of either DNA or RNA pathogens (P>0.05). After liquefaction of simulated sputum samples with 1mol/L NaOH, the MP DNA Ct value significantly increased compared to the trypsin group (t=3.935, P<0.05). No differences in Ct values were observed after neutralization with different concentrations (1, 0.5, and 0.25mol/L) of Tris-HCl (pH=7) (all P>0.05). Conclusions Liquefaction of sputum with 1mol/L NaOH has an inhibitory effect on PCR detection, which may lead to false-negative results. The inhibition can be eliminated to some extentby using Tris-HCl, to improve the accuracy and sensitivity of PCR.

参考文献/References:

[1] 杨晓慧,许蕾,晏耀明,等.多重PCR高分辨熔解分析同时检测骨髓增殖性肿瘤JAK2,MPL及CALR基因突变的初步研究[J].现代检验医学杂志,2023,38 (4):63-66, 71. YANG X H, XU L, YAN Y M, et al. Preliminary study on simultaneous detection of JAK2,MPL and calreticulin mu-tations in patients with myeloproliferative neoplasms by multiplex PCR high resolution melting analysis[J]. Journal of Modern Laboratory Medicine, 2023, 38(4): 63-66, 71.
[2] 李团团,叶成果,李振,等.两种RT-PCR检测SARS-CoV-2核酸试剂盒实验室应用评价[J].现代检验医学杂志,2020,35(3):90-93. LI T T, YE C G, LI Z, et al. Evaluation of laboratory application of two RT-PCR detection kits for SARS-CoV-2 nucleic acid[J]. Journal of Modern Laboratory Medicine, 2020, 35(3): 90-93.
[3] 马涛.哈米拉·玉素甫.检验科微生物检验质量的影响因素及病原菌耐药性调查[J].中文科技期刊数据库:(全文版)医药卫生,2024(3):0020-0023. MA T, HAMILA Y. Investigation on the influencing factors of microbiological testing quality in laboratory and pathogen resistance[J]. Chinese Science and Tech-nology Journal Database (Full Text Edition) Medical and Health, 2024(3): 0020-0023.
[4] 张海邻,李昌崇.《儿童呼吸道感染微生物检验标本采集转运与检测建议(病毒篇)》解读[J].中国实用儿科杂志,2019,34(2):89-93. ZHANG H L, LI C C. Interpretation of advice on the collection, transfer and detection of microbiological testing specimen in children with respiratory infection (focusing on virus) [J]. Chinese Journal of Practical Pe-diatrics, 2019, 34(2): 89-93.
[5] 赵淑凤,樊玉凤.临床微生物标本规范采集和送检在临床中的重要作用[J].世界最新医学信息文摘(连续型电子期刊),2020,20(73):357-358. ZHAO S F, FAN Y F. The important role of standardized col-lection and submission of clinical microbiological specimens in clinical practice[J]. World’s Latest Medical Information (Continuous Electronic Journal), 2020, 20(73): 357-358.
[6] 何慧,陈懿,潘平,等.不同液化处理及核酸共提取方法对痰样本中病毒核酸的提取效果比较[J].国际流行病学传染病学杂志,2016,43(2):81-85. HE H, CHEN Y, PAN P, et al. Effects of different homogeni-zation and nucleic acid co-extraction methods on viral RNA and DNA extraction from sputum[J]. International Journal of Epidemiology and Infectious Disease, 2016, 43(2): 81-85.
[7] ZHAO M C, JIANG Y, LI G X, et al. Simultaneous detec-tion of 9 respiratory pathogens using a newly developed multiplex real-time PCR panel based on an automatic mo-lecular detection and analysis system[J]. Diagnostic Mi-crobiology and Infectious Disease, 2022, 104(4): 115801.
[8] RADER T S 4, STEVENS M P, BEARMAN G. Syndromic multiplex polymerase chain reaction (mPCR) testing and antimicrobial stewardship: current practice and future direc-tions[J]. Current Infectious Disease Reports, 2021, 23(4): 5.
[9] GIR?N P?REZ D A, FONSECA-AG?ERO A, TOLE-DO-IBARRA G A, et al. Post-COVID-19 syndrome in outpatients and its association with viral load[J]. Inter-national Journal of Environmental Research and Public Health, 2022, 19(22): 15145.
[10] 唐月明,伊洁.数字聚合酶链反应(dPCR)技术在病原体基因检测应用中的研究进展[J].现代检验医学杂志,2021,36(5):174-179. TANG Y M, YI J. Recent advances in research on digi-tal polymerase chain reaction(dPCR) in pathogen gene detection[J]. Journal of Modern Laboratory Medicine, 2021, 36(5): 174-179.
[11] 陈晴晴,何军,俞俊岭,等.安徽省COVID-19病例临床标本检测分析[J].中华实验和临床病毒学杂志, 2020,34(4):382-384. CHEN Q Q, HE J, YU J L, et al. Detection and analysis of clinical specimens of COVID-19 in Anhui prov-ince[J]. Chinese Journal of Experimental and Clinical Virology, 2020, 34(4): 382-384.
[12] 牛卫东,段江洋,蒋诗苑,等.咽拭子?鼻拭子?痰液中SARS-CoV-2核酸检测结果一致性及检出率分析[J].传染病信息,2020,33(4):307-311. NIU W D, DUAN J Y, JIANG S Y, et al. Analysis of con-sistency and positive rate of SARS-CoV-2 nucleic acid test results in oropharyngeal swabs, nasal swabs and sputum[J]. Infectious Disease Information, 2020, 33(4): 307-311.
[13] 国家卫生计生委合理用药专家委员会儿童用药专业组. 中国儿童肺炎支原体感染实验室诊断规范和临床实践专家共识(2019年)[J]. 中华儿科杂志, 2020,58(5):366-373. Expert Committee on Rational Use of Medicines for Children Pharmaceutical Group, National Health and Family Planning Commission. Expert consensus on lab-oratory diagnostics and clinical practice of Mycoplasma pneumoniae infection in children in China (2019)[J]. Chinese Journal of Pediatrics, 2020, 58(5): 366-373.
[14] WILKINSON R C, MELDRUM K, MAGGS C J, et al. Determining the efficacy of disinfectants at nucleic acid degradation[J]. Journal of Applied Microbiology, 2023, 134(11): lxad244.
[15] 林敏,农天雷,刘大鹰.胰酶化痰法检测五项肺肿瘤标志物探讨[J].标记免疫分析与临床,2011,18(3):143-145. LIN M, NONG T L, LIU D Y. Detection of five tumor markers in lung cancer by trypsin digestion of sputum method[J]. Labeled Immunoassays and Clinical Medi-cine, 2011, 18(3): 143-145.
[16] 周步全,朱建峰,王迎春,等.结核分枝杆菌标本的不同处理方法对PCR扩增敏感性的影响[J].现代检验医学杂志,2005,20(6):60-63.ZHOU B Q, ZHU J F, WANG Y C, et al. The influence of different processing methods for Mycobacterium tubercu-losis specimens on PCR amplification sensitivity[J]. Jour-nal of Modern Laboratory Medicine, 2005, 20(6): 60-63.
[17] 刘金花,李小玉,罗一钧,等.不同浓度氢氧化钠对结核分枝杆菌罗氏培养检测结果的影响[J].基层医学论坛,2020,24(23):3257-3259. LIU J H, LI X Y, LUO Y J, et al. Effect of different con-centrations of Sodium hydroxide on Roche culture test results of mycobacterium tuberculosis [J]. the Medical Forum, 2020, 24(23): 3257-3259.
[18] 张军力,托娅,王育民.不同的液化方法对痰液DNA提取效果比较[J].检验医学,2009,24(6):456-458. ZHANG J L, TUO Y, WANG Y M. Comparison of different liquefaction methods for extracting sputum DNA[J]. Laboratory Medicine, 2009, 24(6): 456-458.
[19] INGR M, DOST?L J, MAJEROV? T. Enzymological description of multitemplate PCR-shrinking amplification bias by optimizing the polymerase-template ratio[J]. Jour-nal of Theoretical Biology, 2015, 382: 178-186.

备注/Memo

备注/Memo:
基金项目：中央高水平医院临床科研业务费资助（2022-PUMCH-B-028）。
作者简介：赵伟凯（1990-），男，本科，主管检验技师，研究方向：临床分子检验，E-mail：zhaoweikai719@126.com。
通讯作者：伊洁（1983-），女，博士，副研究员，研究方向：临床微生物分子诊断，E-mail：yijie0908@126.com。
更新日期/Last Update: 2026-01-15