Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)–dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA–DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin–streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR–dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR–dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.

甲型流感、乙型流感、严重急性呼吸综合征冠状病毒 2、腺病毒、呼吸道合胞病毒、肺炎支原体和肺炎衣原体是常见的病原体，可引起重症肺炎等症状，导致急性下呼吸道感染。本研究的目的是设计和评估一种灵敏且特异的多重一步逆转录 PCR (RT-PCR)-试纸层析方法，用于同时快速检测这七种病原体。链霉亲和素包被的蓝色乳胶颗粒用于读出阳性信号。基于正向引物的寡核苷酸序列 (Tag) 与试纸上的互补寡核苷酸序列 (cTag) 的 DNA-DNA 杂交以及生物素-链霉亲和素的相互作用，PCR 产物能够在试纸上被视觉照亮。还评估了特异性和检测限 (LOD)。而且，将该方法的临床性能与 896 个样本的 Sanger 测序进行了比较。未发现与其他病原体的交叉反应，证实了该方法的高度特异性。每种测试病原体的 LOD 为 10 拷贝/µL，整个过程不到 40 分钟。使用 896 个样本，敏感性和特异性均不低于 94.5%。阳性预测值高于82.1%，阴性预测值高于99.5%。PCR-试纸色谱法与 Sanger 测序之间的 kappa 值范围为 0.869 至 0.940。总之，我们的一步法 RT-PCR-试纸色谱法是快速检测多重呼吸道病原体的灵敏且特异的工具。证实了该方法的高特异性。每种测试病原体的 LOD 为 10 拷贝/µL，整个过程不到 40 分钟。使用 896 个样本，敏感性和特异性均不低于 94.5%。阳性预测值高于82.1%，阴性预测值高于99.5%。PCR-试纸色谱法与 Sanger 测序之间的 kappa 值范围为 0.869 至 0.940。总之，我们的一步法 RT-PCR-试纸色谱法是快速检测多重呼吸道病原体的灵敏且特异的工具。证实了该方法的高特异性。每种测试病原体的 LOD 为 10 拷贝/µL，整个过程不到 40 分钟。使用 896 个样本，敏感性和特异性均不低于 94.5%。阳性预测值高于82.1%，阴性预测值高于99.5%。PCR-试纸色谱法与 Sanger 测序之间的 kappa 值范围为 0.869 至 0.940。总之，我们的一步法 RT-PCR-试纸色谱法是快速检测多重呼吸道病原体的灵敏且特异的工具。阳性预测值高于82.1%，阴性预测值高于99.5%。PCR-试纸色谱法与 Sanger 测序之间的 kappa 值范围为 0.869 至 0.940。总之，我们的一步法 RT-PCR-试纸色谱法是快速检测多重呼吸道病原体的灵敏且特异的工具。阳性预测值高于82.1%，阴性预测值高于99.5%。PCR-试纸色谱法与 Sanger 测序之间的 kappa 值范围为 0.869 至 0.940。总之，我们的一步法 RT-PCR-试纸色谱法是快速检测多重呼吸道病原体的灵敏且特异的工具。