数字PCR和二代测序技术在脓毒血症患者病原学诊断中的应用价值  

The application value of digital PCR and next-generation sequencing technology in the etiological diagnosis of sepsis patients

作　　者：史健 雷静 刘泽世[1] 熊朝亮 李恬 耿燕[1] Shi Jian;Lei Jing;Liu Zeshi;Xiong Chaoliang;Li Tian;Geng Yan(Department of Laboratory,the Second Affiliated Hospital of Xi′an Jiaotong University,Xi′an 710004,China;College of Medical Technology,Shaanxi University of Chinese Medicine,Xianyang 712000,China)

机构地区：[1]西安交通大学第二附属医院检验科,西安710004 [2]陕西中医药大学医学技术学院,咸阳712000

出　　处：《中华微生物学和免疫学杂志》2025年第3期256-263,共8页Chinese Journal of Microbiology and Immunology

基　　金：陕西省重点研发计划(2023-YBSF-412)。

摘　　要：目的探讨微滴式数字PCR(droplet digital PCR,ddPCR)和二代测序(next-generation sequencing,NGS)技术在脓毒血症患者病原学诊断中的价值,为脓毒血症早期诊断提供参考依据。方法采用回顾性分析研究,收集2023年2—8月西安交通大学第二附属医院重症监护室(intensive care unit,ICU)收治的53例疑似脓毒血症患者的临床资料,采集血液同步进行血培养血培养、ddPCR与NGS检测。结果去除病毒后,血培养阳性率为18.87%(10/53),ddPCR阳性率为47.17%(25/53)、NGS阳性率为41.51%(22/53)。以ddPCR检测范围为参考,ddPCR阳性率为98.11%(52/53),NGS阳性率为84.91%(45/53),两组间差异有统计学意义(P<0.05)。以血培养为参考,ddPCR与NGS敏感度(60.00%vs 70.00%)、特异度(65.11%vs 69.77%)一致性较好。在病原体检出方面,NGS比ddPCR检测范围更广(34种vs 21种),差异具有统计学意义(χ^(2)=55.000,P<0.001)。在耗时方面,血培养平均耗时66.93 h,ddPCR比NGS更快(约4 h vs 20 h)。在抗微生物耐药(antimicrobial resistance,AMR)基因检测方面,有5个耐药菌株经ddPCR与NGS共同检出,经ddPCR检出2种AMR基因,包括bla KPC和mecA,经NGS检出5种AMR基因,除bla KPC外,其余4种在ddPCR检测靶标外检出。结论相比血培养,ddPCR与NGS在脓毒血症病原学诊断中具有良好应用价值,其中ddPCR对病原体检出率更高、耗时更短,NGS检测范围更广,可应用于一些少见菌或常规难以培养的病原体。ObjectiveTo explore the value of droplet digital PCR(ddPCR)and next-generation sequencing(NGS)technology in the pathogenetic diagnosis of sepsis patients,and to provide a reference basis for the early diagnosis of sepsis.MethodsA retrospective analysis was used to collect the clinical data of 53 patients with suspected sepsis admitted to the intensive care unit(ICU)of the Second Affiliated Hospital of Xi'an Jiaotong University from February to August 2023,and the blood was collected for blood culture,ddPCR and NGS detection simultaneously.ResultsAfter excluding viral infections,the blood culture positive rate was 18.87%(10/53),the ddPCR positive rate was 47.17%(25/53),and the NGS positive rate was 41.51%(22/53).When using the ddPCR detection range as a reference,the ddPCR positivity rate was 98.11%(52/53),while the NGS positivity rate was 84.91%(45/53).There was a statistically significant difference in the positivity rate between the two groups(P<0.05).Using blood culture as a reference,the sensitivity(60.00%vs 70.00%)and specificity(65.11%vs 69.77%)of ddPCR and NGS were in good agreement.In terms of pathogen detection,NGS had a wider detection range than ddPCR(34 species vs 21 species),and the difference was statistically significant(χ^(2)=55.000,P<0.001).In terms of time-consumption,blood culture took 66.93 h on average,while ddPCR was faster than NGS(about 4 h vs 20 h).In terms of antimicrobial resistance(AMR)gene detection,five resistant strains were detected by both ddPCR and NGS,ddPCR detected two AMR genes,namely bla KPC and mecA,while NGS detected five AMR genes.Among them,except for bla KPC which was detected outside the target range in ddPCR,the other four AMR genes were also detected by ddPCR.ConclusionsCompared with blood culture,ddPCR and NGS have good application value in the etiological diagnosis of sepsis.Specifically,ddPCR has a higher detection rate of pathogens and takes less time.On the other hand,NGS has a wider detection range,especially for the discovery of some rare bacteria or pathogens

关 键 词：脓毒血症 数字PCR 二代测序 病原体 病原学诊断 

分 类 号：R446.5[医药卫生—诊断学]