环介导等温扩增技术在下呼吸道感染常见病原体检测中的应用
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国家自然科学基金(81572052);江苏省自然科学基金(BK20151178)。


Application of loop-mediated isothermal amplification technique for the detection of common pathogens in lower respiratory tract infections
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    摘要:

    摘要:目的?应用环介导等温扩增技术(LAMP)检测下呼吸道感染患者标本中的13种病原体,为临床快速诊断感染性疾病提供依据。方法?收集疑似下呼吸道感染患者痰标本103例、肺泡灌洗液45例,常规培养分离细菌、真菌,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行细菌鉴定,同时采用LAMP技术检测13种常见病原体(肺炎链球菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、嗜麦芽窄食单胞菌、流感嗜血杆菌、嗜肺军团菌、肺炎衣原体、肺炎支原体、结核分枝杆菌复合群),实时荧光定量PCR(RT-qPCR)检测45例肺泡灌洗液标本中肺炎支原体,LAMP法检出的结核分枝杆菌阳性标本用二代测序(NGS)技术验证。结果?148例标本中,质谱法检出率为42.6%(63/148),分离出病原菌81株;LAMP法检出率为68.2%(101/148),共检测出175种病原体。质谱法和LAMP法对金黄色葡萄球菌、大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌的检出率差异无统计学意义(P>0.05),但LAMP法对肺炎链球菌、耐甲氧西林金黄色葡萄球菌、流感嗜血杆菌和嗜麦芽窄食单胞菌的检出率高于质谱法(P<0.05)。LAMP法从45例肺泡灌洗液中检出26例肺炎支原体,而采用RT-qPCR检出23例。148例样本中,LAMP法检出2例结核分枝杆菌复合群,并使用NGS验证为阳性。结论?与传统细菌培养鉴定相比,LAMP技术具有检测快速、准确、操作简便等优势,可为临床诊疗提供可靠依据。

    Abstract:

    Abstract:?Objective?To detect 13 kinds of pathogens in the specimens of patients with lower respiratory tract infections by the loop-mediated thermostatic amplification(LAMP)technique, and then provide the evidence for the rapid diagnosis of clinical infectious diseases.Methods?A total of 103 sputum samples and 45 alveolar lavage fluid specimens were collected from the patients with suspected lower respiratory infections.Bacteria and fungi were isolated by routine culture and identified by the matrix assisted laser desorption time-of-flight mass spectrometry(MALDI-TOF MS).Meanwhile, 13 kinds of common pathogens, including?Streptococcus?pneumoniae,?Staphylococcus?aureus, methicillin-resistant?Staphylococcus?aureus,?Escherichia?coli,?Klebsiella?pneumoniae,?Pseudomonas?aeruginosa,?Acinetobacter?baumannii,?Stenotrophomonas?maltophilia,?Haemophilus?influenzae,?Legionella?pneumoniae,?Chlamydia?pneumoniae,?Mycoplasma?pneumoniae?and?Mycobacterium?tuberculosis?complex group, were detected by the LAMP.Mycoplasma?pneumoniae?in 45 alveolar lavage fluid samples was detected by real-time fluorescent quantitative PCR(RT-qPCR).The samples with positive?Mycobacterium?tuberculosis?detected by the LAMP were further verified by the next generation sequencing(NGS).Results?The detection rate of pathogens by the MALDI-TOF MS was 42.6%(63/148), and 81 strains of pathogens were isolated.The detection rate of pathogens by the LAMP was 68.2%(101/148), and 175 kinds of pathogens were detected.There was no significant difference in the detection rate of?Staphylococcus?aureus,?Escherichia?coli,?Klebsiella?pneumoniae,?Pseudomonas?aeruginosa?and?Acinetobacter?baumannii?between the MALDI-TOF MS and LAMP(P>0.05), while the detection rates of?Streptococcus?pneumoniae, methicillin-resistant?Staphylococcus?aureus,?Haemophilus?influenzae?and?Stenotrophomonas?maltophilia?detected by the LAMP were significantly higher than those by the MALDI-TOF MS(P<0.05).Twenty-six cases of?Mycoplasma?pneumoniae?were detected from 45 alveolar lavage fluid specimens by the LAMP, while 23 were detected by RT-qPCR.In 148 samples, 2 cases of?Mycobacterium?tuberculosis?complex group were detected by the LAMP, and confirmed as positive by the NGS.Conclusion?Compared with traditional bacterial culture and identification, LAMP technique has advantages of rapidity, accuracy and simple, which can provide reliable evidence for clinical diagnosis and treatment.

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顾锦,郭会,周围,吴玉敏,孙靖,马锦洪,王玉月,史伟峰.环介导等温扩增技术在下呼吸道感染常见病原体检测中的应用[J].临床检验杂志,2021,39(1):12-16

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  • 收稿日期:2020-08-02
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  • 在线发布日期: 2021-03-19
  • 出版日期: 2021-01-28
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