目的 建立并评价从血清中检测曲霉游离DNA的定量PCR方法.方法 合成曲霉28s rDNA基因特异广谱引物和Taqman探针,建立定量PCR检测方法,并进行方法学评价.用建立的方法检测33例患者的GM阳性血清标本.结果 定量PCR方法灵敏度为2拷贝/反应,标准曲线Y=-3.629X+40.187,相关系数R2为0.997 6.定量PCR方法检测33例患者的首次GM阳性血清标本,对诊断侵袭性曲霉感染的敏感性84％(21/25)、特异性87.5％(7/8)、阳性预测值95.5％(21/22)、阴性预测值63.6％(7/11).PCR阳性与PCR阴性病例组间侵袭性曲霉感染诊断率有统计学差异(95.5％ vs 36.4％),PCR阳性与侵袭性曲霉感染诊断有相关性(RR=2.625).结论 血清曲霉游离DNA定量PCR检测有助于侵袭性曲霉感染的早期诊断.
Establishment and clinical application of quantitative PCR for invasive aspergillosis detection in serum
Objective To establish the quantitative PCR detection of serum aspergillus DNA, wondered whether detecting circulating aspergillus DNA in serum sample PCR could help in diagnosing invasive aspergillosis(lA). Methods Panfungal Aspergillus-specific primers and probe described elsewhere were used,then we set up real time PCR detection method (Optimized reaction system, conditions, and evaluated the methodology. We detected 33 cases of first GM- positive serum samples in this method. Results The developed quantitative PCR method showed high sensitivity (2 copies/reaction) and good specificity,the Standard curve Y =-3. 629X+40. 187,correlation coefficient R2 was 0. 9976. Using realtime PCR to process the 33 first GM-positive serum sample,it showed a sensitivity of 84% (21/ 25) .specificity of 87. 5%{7/8},positive and negative predictive value of 95. 5% (21/22) and 63. 6% (7/11) respectively. The IA diagnosis difference between PCR positive and PCR negative patients was significant (P<0. 001 by Fisher,s,exact test). The relative risk for aprobable or possible IA when the GM test was positive was 2. 625 times higher when the PCR result on the same serum sample was also positive. Conclusion Aspergillus serum cell free DNA may be used as an early diagnostic method for invasive aspergillosis.
NETL
NSTL
万方数据
维普
