Performance evaluation of a nucleic acid detection kit for detecting three kinds of candida in lower respiratory tract samples
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摘要: 目的评估1种应用实时荧光PCR法检测念珠菌(包括白色念珠菌、热带念珠菌和光滑念珠菌)核酸试剂的性能是否满足临床应用要求。方法① 应用含白色念珠菌106和108CFU/mL 2种浓度水平的临床混合下呼吸道样本，按照EP15-A3文件5×5精密度设计方案，5 d内每天每种浓度做5个复孔，共计25孔，提取核酸后应用该试剂扩增以评价其重复性和实验室内精密度；②应用白色念珠菌、热带念珠菌和光滑念珠菌3种念珠菌标准株及大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄色葡萄球菌标准株菌液，提取核酸后应用该试剂扩增以评价其特异性；③将白色念珠菌标准株用生理盐水10倍梯度稀释为1×105、1×104、1×103和1×102CFU/mL，5 d内每天每种浓度4个复孔，共计20个复孔，提取核酸后应用该试剂扩增以评价其检出限；④收集101例来自呼吸系统疾病患者的下呼吸道标本，提取核酸后应用该试剂扩增检测，并与真菌培养法的结果进行比较，以评价其符合率。结果① 该试剂检测含白色念珠菌108CFU/mL和106CFU/mL的临床混合样本的重复性精密度分别为2.18%和1.32%，实验室内精密度分别为2.18%和1.36%；②该试剂检测白色念珠菌、热带念珠菌和光滑念珠菌标准株的结果均为阳性，检测大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄色葡萄球菌标准株的结果均为阴性，无交叉反应；③该试剂检测1×105、1×104、1×103和1×102CFU/mL的白色念珠菌标准株的检出率分别100.00%(20/20)、100.00%(20/20)、65.00%(13/20)和25.00%(5/20)，检出限为5.8×103(95%CI 2.3×103~4.3×104)CFU/mL；④101例下呼吸道标本，该试剂检出的阳性率为79.21%(80/101)，真菌培养法的阳性率为58.42%(59/101)，2者间差异有统计学意义(P＜0.05)；2种方法检测结果一致性中等(Κappa=0.538 8)，阳性符合率为100.00%(59/59)，阴性符合率为50.00%(21/42)。结论该试剂的精密度高、特异性好，与真菌培养的一致性中等，能基本满足临床应用要求。Abstract: ObjectiveTo evaluate the performance of the nucleic acid detection kit for three kinds of Candida including Candida albicans, Candida tropicalis and Candida glabrata using real-time fluorescent polymerase chain reaction(PCR), and assess whether it meets clinical needs.Methods① The clinical mixed samples with high and low concentrations of Candida albicans(108CFU/ml and 106CFU/mL) were extracted and tested five times per day in a single run for five days using the kit, and the repeatability and within-laboratory precision were calculated following EP15-A3. ②The standard strains of Candida Albicans, Candida tropicalis, Candida glabrata, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter baumannii and Staphylococcus aureus were used to verify the specificity of the kit. ③The 1×105, 1×104, 1×103and 1×102CFU/mL of Candida albicans standard strain were extracted and tested four times per day in a single run for five days using the kit. The positive rate of each dilution was counted and the limit of detection(LoD) was evaluated. ④The 101 lower respiratory tract samples from the patients with respiratory diseases were collected, and detected by both real-time fluorescent PCR and fungal culture. The coincidence rate of both assays was analyzed.Results① The repeatability and within-laboratory precision of the kit were 2.18% and 2.18% at 108CFU/mL, and 1.32% and 1.36% at 106CFU/mL, respectively. ②The standard strains of Candida albicans, Candida tropicalis and Candida glabrata were tested positive using the kit, and the standard strains of the other five bacteria including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter baumannii and Staphylococcus aureus were tested negative by the kit. ③The detection rates of Candida albicans standard strain with different concentration were 100.00%(20/20) for 1×105CFU/mL, 100.00%(20/20) for 1×104CFU/mL, 65.00%(13/20) for 1×103CFU/mL and 25.00%(5/20) for 1×102CFU/mL using the kit, respectively. The limit of detection(LoD) was 5.8×103(95%CI 2.3×103-4.3×104) CFU/mL. ④We evaluated the concordance between real-time fluorescent PCR and fungal culture in the 101 lower respiratory tract samples. The results were 80(79.21%) positive and 21(20.79%) negative detected by real-time fluorescent PCR, and 59(58.42%) positive and 42(41.58%) negative detected by fungal culture method, respectively. There was significant difference between the qualitative results detected by both assays(P < 0.05). The moderate agreement was observed between both assays(Κappa=0.538 8). The positive percent agreement and the negative percent agreement were 100.00%(59/59) and 50.00%(21/42) respectively. The overall agreement was 79.21%(80/101).ConclusionThe nucleic acid detection kit of three kinds of Candida had good performance with high precision and good specificity, and exhibited moderate agreement with fungal culture, and could basically meet the clinical needs.
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表 1 念珠菌核酸检测试剂的精密度评价结果
浓度水平/(CFU·mL-1) 重复性精密度 实验室内精密度 X±S CV/% X±S CV/% 106 24.84±0.33 1.32 24.84±0.34 1.36 108 18.28±0.40 2.18 18.28±0.40 2.18 
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