Development and Evaluation of the application effect of PCR assay for detection of Corynebacterium striatum
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摘要:
目的 评价一种快速检测纹带棒杆菌的PCR方法在纹带棒杆菌临床分离株和临床痰标本中的应用效果。 方法 采用针对纹带棒杆菌种特异性基因ftr1设计的PCR引物Cst_1-F/Cst_1-R建立检测纹带棒杆菌的单重PCR方法。 使用纹带棒杆菌标准菌株ATCC6940、ATCC43751、152株纹带棒杆菌临床分离株和30株非纹带棒杆菌菌株验证该方法的有效性，并比较该方法与16S rRNA测序和MALDI-TOF MS鉴定结果的一致性。 对该方法的特异性和敏感性进行评估，最终将该方法用于检测88份临床痰标本。 结果 该PCR方法与16S rRNA测序和MALDI-TOF MS 在鉴定152株纹带棒杆菌临床分离株中具有100%的一致性；该方法对30株非纹带棒杆菌菌株的检测结果均为阴性，检测下限为1 ng模板DNA/反应；在应用于临床痰标本时，该方法与痰涂片镜检相比，具有86.4%（76/88）的一致性。 结论 本研究中的PCR方法具有良好的特异性和敏感性，可替代16S rRNA测序和MALDI-TOF MS成为临床鉴定纹带棒杆菌的便捷、经济、有效的方法，且在检测临床痰标本中的纹带棒杆菌时具有较好的应用效果。 Abstract:Objective To evaluate the application effect of a rapid PCR method for detecting Corynebacterium striatum (C. striatum) in clinical isolates and clinical sputum specimens. Methods A single PCR method for the detection of C. striatum was established using PCR primers Cst_1-F/Cst_1-R designed for the species-specific gene ftr1. The C. striatum reference strain ATCC6940, ATCC43751, 152 clinical isolates and 30 non-C. striatum strains was used to verify the effectiveness of this method. The resultof this PCR method was compared with that of 16S rRNA gene sequencing and MALDI-TOF MS.The specificity and sensitivity of this PCR method were evaluated. Finally, this PCR method was used to detect 88 clinical sputum specimens. Results The result of the PCR method was 100% consistent with that of 16S rRNA sequencing and MALDI-TOF MS in the identification of 152 C. striatum clinical isolates. There was no cross reaction in 30 non-C. striatum strains.The limit of detection was 1 ng template DNA per reaction.When applied this PCR method to detect clinical sputum specimens, the consistency of this method compared with sputum smear microscopy was 86.4% (76/88). Conclusion As a convenient, economic and effective method, the PCR assay in this study may replace 16S rRNA gene sequencing and MALDI-TOF MS for the clinical identification of C. striatum. Also, this assay has good application effect in the detection of C. striatum in clinical sputum specimens. -
Key words:
- Corynebacterium striatum /
- Polymerase chain reaction /
- Identification /
- Detection /
- Application
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图 1 PCR方法的特异性检测
注：M. DL 2000 DNA Marker；1～10. 纹带棒杆菌临床分离株；11. 丙酸棒杆菌；12. 模拟棒杆菌；13. 溶血性葡萄球菌；14. 表皮葡萄球菌；15. 肺炎链球菌；16. 大肠埃希菌；17. 肠炎沙门菌；18. 猪链球菌；19. 艰难梭菌；20. 弗氏枸橼酸杆菌；21. 纹带棒杆菌参考菌株ATCC6940；22. 纹带棒杆菌参考菌株ATCC43751；23. 阴性对照
Figure 1. Specificity test of the PCR method
图 2 PCR方法的敏感性检测
注：M. DL 2000 DNA Marker；1～6. 纹带棒杆菌参考菌株ATCC6940 模板DNA依次为100 ng、10 ng、1 ng、100 pg、10 pg、1 pg；7. 阴性对照
Figure 2. Sensitivity test of the PCR method
图 3 PCR方法应用于88份临床痰标本中纹带棒杆菌的检测
注：M. DL 2000 DNA Marker；1～88. 痰标本；P. 阳性对照；N. 阴性对照
Figure 3. Detection ofCorynebacterium striatum in 88 clinical sputum specimens using PCR assay
表 1 本研究所用菌株信息
Table 1. Strains used in this study
菌种名称 菌株编号 菌株数量 纹带棒杆菌 ATCC6940;
ATCC437512 分离株 152 模拟棒杆菌 分离株 1 丙酸棒杆菌 分离株 1 鲍曼不动杆菌 分离株 1 洋葱伯克霍尔德菌 分离株 1 弗氏枸橼酸杆菌 ATCC43864; ATCC8090 2 布氏枸橼酸杆菌 分离株 1 杨氏枸橼酸杆菌 分离株 1 艰难梭菌 ATCC BAA-1803 1 粪肠球菌 分离株 1 大肠埃希菌 ATCC25922 1 肺炎克雷伯菌 分离株 1 鼻硬结克雷伯菌 分离株 1 单增李斯特菌 EDG-e 1 摩氏摩根菌 分离株 1 类志贺邻单胞菌 分离株 1 铜绿假单胞菌 ATCC15442 1 肠炎沙门菌 分离株 1 粘质沙雷菌 分离株 1 痢疾志贺菌 分离株 1 福氏志贺菌 分离株 1 金黄色葡萄球菌 分离株 1 表皮葡萄球菌 分离株 1 溶血性葡萄球菌 分离株 1 牛链球菌 分离株 1 肺炎链球菌 ATCC49619 1 酿脓链球菌 分离株 1 血链球菌 分离株 1 猪链球菌 GZ1 1 小肠结肠炎耶尔森菌 分离株 1 
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