Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, PR China.
Clin Chem Lab Med. 2010 Oct;48(10):1501-5. doi: 10.1515/CCLM.2010.296. Epub 2010 Jul 27.
Accurate and early diagnosis of tuberculosis (TB) is of major importance in the management and control of TB. Because the conventional bacteriological diagnosis of TB has several limitations, nucleic acid amplification (NAA) tests have emerged as promising alternatives. A potential problem with NAA tests is that some strains lack a target, which may be the one of main reasons for the much lower and highly variable accuracy in diagnosis. A possible solution may be to use more valid and applicable targets to increase detection accuracy.
In this paper, we designed a two-step program to obtain NAA test targets. Inter-simple sequence repeats (ISSR) based on oligonucleotide (GTG)(5) were first constructed to genotype Mycobacterium strains to obtain Mycobacterium tuberculosis (MTB)-specific fragment. Second, sequence characterized amplified region (SCAR) markers were developed from these species-specific sequences to identify MTB. Some 312 Mycobacterium strains were used to evaluate the efficacy of the SCAR markers, IS6110 element [specific identification of Mycobacterium tuberculosis complex (MTC)] and 16SrRNA gene (specific identification of Mycobacterium) amplification, together with traditional bacteriology testing was used as a control.
MTB-specific sequences located in a gene coding for Rv1508c, as a new NAA test target, were obtained using ISSR-PCR genotyping. Based on these sequences, the SCAR primer pairs MISP1 and MISP2 were designed. All 312 strains from Mycobacterium accurately produced the genus-specific 16SrRNA amplicon. 271 MTB strains and M. africanum were positive. However, all nontuberculous mycobacteria (NTM) strains and 1 MTB strain named 1143 were negative in both SCAR and IS6110 PCR amplification. M. bovis, bacille Calmette-Guérin (BCG) were IS6110-PCR positive, while SCAR-PCR was negative. Strain 1143 was defined as M. arupense with 99% identity by 16SrRNA gene sequencing identification, despite being diagnosed as MTB using traditional testing.
SCAR markers developed with this two-step program can be used as a new NAA test target to correctly detect MTB.
准确和早期诊断结核病（TB）在结核病的管理和控制中非常重要。由于传统的细菌学诊断 TB 有几个局限性，核酸扩增（NAA）检测已经成为有前途的替代方法。NAA 检测的一个潜在问题是，一些菌株缺乏靶标，这可能是诊断准确性低且高度可变的主要原因之一。一个可能的解决方案可能是使用更有效和适用的靶标来提高检测的准确性。
在本文中，我们设计了一个两步程序来获得 NAA 检测靶标。首先，基于寡核苷酸（GTG）（5）的简单重复序列间（ISSR）构建来对分枝杆菌菌株进行基因分型，以获得结核分枝杆菌（MTB）特异性片段。其次，从这些种特异性序列中开发序列特征扩增区（SCAR）标记，以鉴定 MTB。使用 312 株分枝杆菌菌株评估 SCAR 标记、IS6110 元件（MTB 复合群的特异性鉴定）和 16SrRNA 基因（分枝杆菌的特异性鉴定）扩增的功效，同时使用传统细菌学检测作为对照。
使用 ISSR-PCR 基因分型获得了位于 Rv1508c 基因中、作为新的 NAA 检测靶标的 MTB 特异性序列。基于这些序列，设计了 SCAR 引物对 MISP1 和 MISP2。来自分枝杆菌的所有 312 株菌株都准确地产生了属特异性 16SrRNA 扩增子。271 株 MTB 株和 M. africanum 为阳性。然而，所有非结核分枝杆菌（NTM）株和 1 株命名为 1143 的 MTB 株在 SCAR 和 IS6110 PCR 扩增中均为阴性。M. bovis、卡介苗（BCG）是 IS6110-PCR 阳性，而 SCAR-PCR 为阴性。尽管传统检测将菌株 1143 诊断为 MTB，但通过 16SrRNA 基因测序鉴定，其被定义为 M. arupense，同源性为 99%。
使用此两步程序开发的 SCAR 标记可作为新的 NAA 检测靶标，用于正确检测 MTB。
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