荧光定量PCR检测抗酸染色涂片刮削物中结核分枝杆菌DNA的可行性

The feasibility of detecting mycobacterium tuberculosis DNA from scraping things of acid-fast stained smear slides by fluorescence quantitative PCR

目的:分析荧光定量PCR技术检测抗酸染色涂片刮削物中结核分枝杆菌DNA的检测能力。方法:应用结核分枝杆菌(TB)核酸检测试剂(PCR-荧光探针法)检测80例阴性、25例1-9条/300视野、95例1+、40例2+、21例3+、20例4+抗酸染色显微镜检查结果的涂片刮削物结核分枝杆菌DNA,分析不同阳性等级涂片的靶DNA检出情况,并应用Spearman等级相关分析阳性等级与检出基因拷贝数的相关性。结果:在1-9条/300视野、1+、2+、3+、4+的涂片刮削物中,分别有7、48、31、19、20例结核分枝杆菌DNA检测阳性,阳性检出率分别为28.00%、50.53%、77.50%、90.48%和100.00%,总检出率为62.19%,阳性等级与靶基因拷贝数检测结果呈正相关(r=0.684,P<0.001);80例阴性涂片刮削物检测结果均呈阴性。结论:荧光定量PCR技术对2+以上的抗酸染色涂片刮削物中的结核分枝杆菌DNA具有较好的检测效能,但对本属于荧光定量PCR技术检测灵敏度范围的1+涂片的低阳性检出率则需更进一步的研究。

Objective To analyze the feasibility of detecting Mycobacterium tuberculosis DNA from scraping things of acid-fast stained smear slides by fluorescence quantitative PCR. Methods 281 acid-fast-stained smear slides [ including 80 negative slides marked as -, 25 slides with a score of 1-9 bacteria per 300 microscope fields （marked as ±）, 95 slides with a score of 1＋, 40 slides with a score of 2＋, 21 slides with a score of 3＋, and 20 slides with a score of 4＋ according to acid-fast-stained smear microscopy procedures ] were collected, and their mycobactefium tuberculosis DNA from scraping things of stained smear slides were detected by fluorescence quantitative PCR. Then the detecting results were analyzed in accordance with positive degree of acid-fast-stained smear, and the correlation between positive degree of acid-fast stained smear and target DNA copies was studied by Spearman rank correlation. Results Of these scraping things of stained smear slides from ± slides to 4＋ slides in order, 7, 48, 31, 19, and 20 cases were positive respectively for mycobaeterium tuberculosis DNA detection. The positive rates were 28.00%, 50.53%, 77.50%, 90.48%, and 100.00%, respectively, whilst it was 62.19% for all these samples. A positive correlation between positive degree of acid-fast-stained smear and target DNA copies was found （r = 0.684, P 〈 0.001 ）. As for scraping things of 80 negative slides, none of them was positive for mycobacterium tuberculosis DNA detection. Conclusion Fluorescence quantitative PCR is effective in detecting mycobactefium tuberculosis DNA from scraping things of acid-fast stained smear slides above 2＋. But for smear with a score of 1 ＋, it should be studied further why presented very lower positive rate than theory prediction as bacteria number in it is within the sensitivity range of fluorescence quantitative PCR technique theoretically.