【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1×10~6copies/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/L NaOH，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/L NaOH液化处理。模拟痰液样本用1mol/L NaOH液化后，加不同浓度三羟甲基氨基甲烷盐酸盐[Tris(hydroxymethyl) aminomethane hydrochloride,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(polymerase chain reaction,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/L NaOH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/L NaOH液化处理对2 000和1 000 copies/ml MP DNA无影响(t=0.946、1.925，均P>0.05)，但对低载量(200和500 copies/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/L NaOH液化后，MP DNA Ct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0.25 mol/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/L NaOH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。更多还原

【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 1×10~6 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), 1 mol/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 1 mol/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 0.25 mol/L) of Tris-HCl(pH=7)(all P>0.05). Conclusions Liquefaction of sputum with 1 mol/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.更多还原