酶促重组等温扩增结合的胶体金侧流层析检测鹦鹉热衣原体

Detection of Chlamydia psittaci by enzymatic recombinase amplification coupled with lateral flow immunochromatographic assay

摘要:

【背景】鹦鹉热衣原体(Chlamydia psittaci)是一种重要的人畜共患病病原体，可引起鹦鹉热，该病若未能得到及时诊断和治疗，将会危及患者生命健康。【目的】建立一种基于酶促重组等温扩增(enzymatic recombinase amplification,ERA)结合胶体金侧流层析技术的快速检测鹦鹉热衣原体的方法。【方法】以鹦鹉热衣原体主要外膜蛋白(major outer membrane protein,MOMP)基因保守序列为靶标，设计特异性引物和探针。通过优化反应体系中的探针浓度、孵育温度和反应时间等关键参数，建立ERA结合胶体金侧流层析检测方法。评估该方法的灵敏度与特异性，并使用49份临床生物样本进行方法验证。【结果】该方法在37℃条件下15 min内可检测出最低10 copies/μL的鹦鹉热衣原体DNA。特异性测试表明，该方法仅对鹦鹉热衣原体呈阳性反应，对其他4种相关呼吸道病原体均无交叉反应。相较于实时荧光定量PCR (real time fluorescence quantitative PCR,RT-qPCR)方法，该方法在49份临床样本的检验中灵敏度为94.7%(36/38)、特异度为100%(11/11)、阴性预测值为84.6%(11/13)、阳性预测值为100%(36/36)、Kappa值为0.89。结果显示该方法具有良好的临床应用前景。【结论】本研究建立的ERA结合胶体金侧流层析方法操作简便、无需特殊仪器设备，能够快速、灵敏地检测鹦鹉热衣原体。该方法为鹦鹉热衣原体的快速诊断提供了新的技术支持，适用于资源有限的实验室和现场检测。

Abstract:

[Background] Chlamydia psittaci is the causative agent of parrot fever, a zoonotic infectious disease. Without timely diagnosis and appropriate treatment, parrot fever can pose a serious threat to patients’ lives.[Objective] To develop a rapid and sensitive method for detecting C. psittaci by enzymatic recombinase amplification (ERA) in conjunction with lateral flow immunochromatographic assay. [Methods] Specific primers and probes were designed targeting the conserved sequence of the major outer membrane protein (MOMP) gene of C. psittaci. The reaction system was optimized by adjusting key parameters such as probe concentration, incubation temperature, and reaction time to establish the detection method based on ERA coupled with lateral flow immunochromatographic assay. The sensitivity and specificity of the method were evaluated, and then the method was used to detect 49 clinical biological samples. [Results] Under optimal conditions at 37 ℃, the established method could detect C. psittaci DNA at a concentration as low as 10 copies/μL within 15 min. The established method showed positive results exclusively for C. psittaci, with no cross-reactivity observed among four other related pathogens. In comparison with the RT-qPCR results for the 49 clinical samples, the method established in this study exhibited the sensitivity of 94.7% (36/38), the specificity of 100% (11/11), the negative predictive value of 84.6% (11/13), the positive predictive value of 100% (36/36), and the Kappa value of 0.89. The results suggested that this method held promising prospects for clinical application. [Conclusion] The ERA coupled with lateral flow immunochromatographic assay established in this study enables rapid and sensitive detection of C. psittaci, featuring ease of operation and no need for specialized equipment. This method provides new technical support for the rapid diagnosis of C. psittaci, especially suitable for resource-limited laboratories and field testing.