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目的了解海南省不明原因发热病人中斑点热群立克次体的感染情况和流行病学特点,分析立克次体病实验检测方法的特点,拟建立一种快速、特异性强、灵敏度高、重复性好的实验室检测技术,为斑点热的防控提供科学依据。方法分别采用巢式聚合酶链式反应(polymerase chain reaction,PCR)、多重实时荧光定量PCR、间接免疫荧光法和病原学分离培养法对不明原因发热病人血标本进行检测。结果用巢式PCR检测发热病人399份血标本中的gro EL基因的特异性片段,检测出17份斑点热群及斑疹伤寒群立克次体阳性,61份恙虫病东方体阳性。用多重实时荧光定量PCR检测出2份斑点热群立克次体阳性,6份斑疹伤寒群立克次体阳性,49份恙虫病东方体阳性。用间接免疫荧光实验检测,斑点热群立克次体Ig M阳性结果为19份,Ig G为148份;斑疹伤寒群立克次体Ig M阳性结果为29份,Ig G为169份。采用L929单层细胞对血标本进行病原学分离培养,培养结果为阴性。结论四种实验室检测方法从病原学、血清学和分子生物学方面进一步阐明斑点热的自然疫源性特点,为其诊断及防护提供有力的实验室依据。
Objective To understand the infection and epidemiological characteristics of Rickettsia plague in Hickman’s disease of unknown origin in Hainan Province and to analyze the characteristics of Rickettsia disease experimental detection method and to establish a rapid, specific, sensitive, Repeatability of laboratory testing technology for the prevention and control of speck fever to provide a scientific basis. Methods The blood samples of patients with unexplained fever were detected by nested polymerase chain reaction (PCR), multiplex real-time quantitative PCR, indirect immunofluorescence and etiological isolation and culture methods, respectively. Results The nested PCR was used to detect the specific fragments of gro EL gene in 399 blood samples of fever patients. 17 positive spots of typhus fever and typhus were identified, and 61 were positive for tsutsugamushi. Rickettsia polygamist was positive for 2 copies of RAPD, Rickettsiae positive for 6 typhus, and Roche positive for 49 tsutsugamushi. Indirect immunofluorescence assay showed that IgM positive results were 19 and Ig G were positive for spot fever group Rickettsia IgM. 29 positive IgM IgM and 169 Ig Ig were positive for typhus. L929 monolayer cells were used to isolate and culture the blood samples. The result of culture was negative. Conclusion The four laboratory tests further elucidate the natural foci of spot fever from the aspects of etiology, serology and molecular biology, and provide a powerful laboratory evidence for their diagnosis and protection.