两步聚合酶链反应法快速诊断感染性角膜炎和眼内炎病原体的研究

Rapid pathogens diagnosis of infected keratitis and endophthalmitis using two steps polymerase chain reaction

目的 探讨两步聚合酶链反应 (PCR)技术快速检测感染性角膜炎和眼内炎病原体的临床诊断效果。方法 采集 2 0 0 1年 9月至 2 0 0 2年 5月 5 1例感染性角膜炎患者的角膜刮片或泪液和 16例眼内炎的玻璃体液 ,先用多重聚合酶链反应 (MPCR)技术检测病原体是细菌、真菌还是单纯疱疹病毒 ,然后用特异性聚合酶链反应 (SPCR)检测病原体细菌或真菌的种类。比较PCR与微生物培养所需的时间和阳性率及其与临床诊断的符合率。结果 6 7例标本经MPCR检测 ,有 5 2例 (77 6 % )阳性 (8例为两种病原体阳性 )。其中 ,细菌阳性的 2 8例经SPCR检测 ,有 7例 (2 5 0 % )为金黄色葡萄球菌、肺炎链球菌或铜绿假单胞菌 ;真菌阳性的 15例经SPCR检测 ,有 8例 (5 3 3% )为念珠菌、曲霉菌或镰刀菌。微生物培养 2 1例 (31 3% )阳性 (1例为两种病原体阳性 )。MPCR检测细菌和真菌的阳性率 (5 8 2 % )高于微生物培养的阳性率 (χ2 =13 5 0 ,P <0 0 0 5 )。微生物培养时间需 72～ 96h ,两步PCR扩增只需 8h。临床诊断的感染性角膜炎与实验室检测符合率为 5 4 8%。结论两步PCR法检测病原体具有速度快、阳性率高等优点 ,有助于感染性眼病的快速病因诊断。

Objective To establish a rapid diagnosis technique of the pathogens of infected keratitis and endophthalmitis.Methods Fifty-one corneal scraping ,tear and 16 vitreous specimens were obtained from 67 cases of infected keratitis and endophthalmitis . Multiplex polymerase chain reaction (MPCR) was performed to differentiate the genes of bacteria, fungi and HSV using their own universal primers. Special-primers PCR were carried out for MPCR positive specimens to detect the genes of common bacteria and fungi respectively. The results were compared with microbe culture and clinical diagnosis.Results In 67 clinical samples, 52(77.6%) were positive at least one kind of pathogen by MPCR( two positive pathogens were 8 cases).SPCR revealed that 7 bacteria (25.0%) out of 28 bacteria positive specimens were Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa. Eight fungi (53.3%)of 15 fungi positive specimens was Candida, Aspergillus, Fusarium. The culture positive rates were 31.3% (21 cases), among them 1 case was found positive for two pathogens. MPCR positive rate was statistically higher than the rate that in culture (χ2=13.50,P<0.005). The kinds of bacteria and fungi of SPCR positive were as the same as bacteria and fungi of culture positive. Consistency of clinical diagnosis and experimental evidences of infected keratitis was 54.8%. The time for microbe culture needs 72 to 96 hours, but MPCR and SPCR needs 8 hours.Conclusion Two steps PCR is rapid, sensitive method to detect the pathogens of infected keratitis and endophthalmitis.