Kulski J K, Khinsoe C, Pryce T, Christiansen K
Department of Microbiology, Royal Perth Hospital, Australia.
J Clin Microbiol. 1995 Mar;33(3):668-74. doi: 10.1128/jcm.33.3.668-674.1995.
The presence of mycobacteria in blood culture fluids (BACTEC) of AIDS patients with positive growth indices (GIs, > 20 U) was investigated by using a multiplex PCR to detect and identify members of the genus Mycobacterium, M. avium, M. intracellulare, and M. tuberculosis. Three different methods of extracting mycobacterial DNA from blood culture fluid were compared for use with the multiplex PCR. Mycobacterial cells were pelleted from a small aliquot of blood culture fluid by centrifugation, and the DNA was extracted from cells by heat lysis or a sodium iodide-isopropanol or a phenol-chloroform method. DNAs of different sizes were amplified from a region of the MPB70 gene of M. tuberculosis (372 bp) and from a region of the 16S rRNA gene of members of the genus Mycobacterium (1,030 bp), M. intracellulare (850 bp), or M. avium (180 bp) as a multiplex PCR in a single tube. The amplified DNA products were detected by agarose gel electrophoresis and ethidium bromide staining in all 41 (100%) positive cultures after sodium iodide-isopropanol extraction, in 18 (44%) after heat lysis, and in 5 (12%) after phenol-chloroform extraction. Of the 41 positive cultures, 38 were identified as M. avium and 2 were identified as M. intracellulare by both routine methods and multiplex PCR. The remaining mycobacterium was identified as M. intracellulare by routine methods and as M. avium by the multiplex PCR. Another six blood cultures that were negative for the presence of acid-fast bacilli after Ziehl-Neelson staining were also negative by PCR. The study shows that the multiplex PCR is a useful method for the detection and identification of either M. avium or M. intracellulare in small samples of cultured BACTEC 13A fluid with positive GIs ranging from 21 to 999 U. The average time to positive GI was 18 days (median, 13 days) and ranged between 8 and 42 days. The multiplex PCR may permit cultured mycobacteria to be identified at an earlier stage than the routine methods which have been adapted for use with the BACTEC system. The results also show that the method selected for extracting mycobacterial DNA from blood culture fluids is crucial for providing sensitive and accurate PCR results.
采用多重聚合酶链反应（PCR）检测和鉴定分枝杆菌属、鸟分枝杆菌、胞内分枝杆菌和结核分枝杆菌，以研究生长指数（GI，>20 U）为阳性的艾滋病患者血培养液（BACTEC）中分枝杆菌的存在情况。比较了三种从血培养液中提取分枝杆菌DNA的不同方法，用于多重PCR。通过离心从小份血培养液中沉淀分枝杆菌细胞，然后通过热裂解、碘化钠 - 异丙醇法或酚 - 氯仿法从细胞中提取DNA。在单管中进行多重PCR，从结核分枝杆菌MPB70基因区域（372 bp）以及分枝杆菌属、胞内分枝杆菌（850 bp）或鸟分枝杆菌（180 bp）的16S rRNA基因区域扩增不同大小的DNA。经碘化钠 - 异丙醇提取后，所有41份（100%）阳性培养物中的扩增DNA产物通过琼脂糖凝胶电泳和溴化乙锭染色检测到；热裂解后，18份（44%）检测到；酚 - 氯仿提取后，5份（12%）检测到。在41份阳性培养物中，通过常规方法和多重PCR鉴定出38份为鸟分枝杆菌，2份为胞内分枝杆菌。其余分枝杆菌通过常规方法鉴定为胞内分枝杆菌，通过多重PCR鉴定为鸟分枝杆菌。另外6份经萋 - 尼染色未发现抗酸杆菌的血培养物，PCR检测也为阴性。该研究表明，多重PCR是一种用于检测和鉴定培养的BACTEC 13A培养液小样本中鸟分枝杆菌或胞内分枝杆菌的有用方法，这些样本的阳性GI范围为21至999 U。阳性GI的平均时间为18天（中位数为13天），范围在8至42天之间。与已适用于BACTEC系统的常规方法相比，多重PCR可能使培养的分枝杆菌在更早阶段得到鉴定。结果还表明，从血培养液中提取分枝杆菌DNA所选择的方法对于提供灵敏和准确的PCR结果至关重要。
