PDF(Pubmed)
Abstract：
UNASSIGNED: The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance.
UNASSIGNED: Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison.
UNASSIGNED: The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%.
UNASSIGNED: MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.
UNASSIGNED: Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison.
UNASSIGNED: The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%.
UNASSIGNED: MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.
摘要：
■世界卫生组织（WHO）《2021年全球结核病报告》指出，利福平耐药结核病（RR-TB）仍然是主要的公共卫生威胁。然而,实践中的RR-TB诊断技术有多种限制，包括更长的时间，缺乏敏感性,且检测不到低比例的异质耐药。
■在这里，我们开发了一种基于多重LNA探针的RAP方法（MLP-RAP），用于更灵敏地检测RR-TB的多点突变及其异质抗性。从国家结核病参考实验室收集的126个临床分离株和78个痰样本，中国疾控中心,通过MLP-RAP检测。并行,还进行巢式PCR产物测定的qPCR和Sanger测序以进行比较。
■使用重组质粒，MLP-RAP测定的灵敏度可以达到5拷贝/μl，比qPCR(100拷贝/μl)灵敏20倍。此外,对利福平异质耐药的检测能力为5%。MLP-RAP测定对核酸提取的要求低(煮沸法),并且当置于荧光qPCR仪器中时,反应可以在1小时内完成。临床评价结果表明,MLP-RAP方法可以覆盖516、526、531和533个密码子,具有良好的特异性。经MLP-RAP检测，78份煮沸痰标本中有41份呈阳性，通过巢式PCR产物测定的Sanger测序进一步证实了这一点，相反,qPCR仅能够检测32个样品。与Sanger测序的巢式PCR产物法比较,MLP-RAP检测的特异性和敏感性均为100%.
■MLP-RAP测定可以检测RR-TB感染，具有很高的敏感性和特异性，表明该方法具有应用于一般实验室的快速和灵敏的RR-TB检测的前景。
■在这里，我们开发了一种基于多重LNA探针的RAP方法（MLP-RAP），用于更灵敏地检测RR-TB的多点突变及其异质抗性。从国家结核病参考实验室收集的126个临床分离株和78个痰样本，中国疾控中心,通过MLP-RAP检测。并行,还进行巢式PCR产物测定的qPCR和Sanger测序以进行比较。
■使用重组质粒，MLP-RAP测定的灵敏度可以达到5拷贝/μl，比qPCR(100拷贝/μl)灵敏20倍。此外,对利福平异质耐药的检测能力为5%。MLP-RAP测定对核酸提取的要求低(煮沸法),并且当置于荧光qPCR仪器中时,反应可以在1小时内完成。临床评价结果表明,MLP-RAP方法可以覆盖516、526、531和533个密码子,具有良好的特异性。经MLP-RAP检测，78份煮沸痰标本中有41份呈阳性，通过巢式PCR产物测定的Sanger测序进一步证实了这一点，相反,qPCR仅能够检测32个样品。与Sanger测序的巢式PCR产物法比较,MLP-RAP检测的特异性和敏感性均为100%.
■MLP-RAP测定可以检测RR-TB感染，具有很高的敏感性和特异性，表明该方法具有应用于一般实验室的快速和灵敏的RR-TB检测的前景。
