Establishment of a cross primer isothermal amplification technique for rapid detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria

doi:10.19982/j.issn.1000-6621.20230088

Abstract

Abstract:

Objective: To establish a molecular cross primer isothermal amplification (CPA) detection system for rapid detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Methods: Six sets of CPA primers and three probes were designed for 16S rRNA-a highly conservative gene sequence of mycobacterium. Using a plasmid containing 16S rRNA gene sequence (with a concentration of 1000 copies/μl) as a positive template, the optimal primer probe combination was selected, and the sensitivity, specificity, and inclusiveness of the established optimal reaction system were analyzed. Samples of 125 presumptive tuberculosis patients with signs or symptoms of tuberculosis from the Second Hospital of Longyan, Fujian Province from February 8, 2022 to March 14, 2023, were collected and then detected by the mycobacterium nucleic acid detection kit (PCR-fluorescence probe method) and the established CPA method. The consistency of the test results between the two methods was statistically analyzed, setting Kappa value ≥0.75 as having good consistency. Results: MTBC/NTM-16S-6 primer and MTBC/NTM-16S-LR-FAM-3 were selected as the best primer probe combinations for the mycobacterium detection system. The minimum detection limit of the detection system was 100 copies/μl, and the detection time was 40 minutes. It could detect MTBC and clinically common NTM, and show no cross reaction with other common respiratory tract bacteria. Compared with the real-time fluorescence quantitative PCR (RT-PCR) detection method, the positive prediction value of the established CPA system was 95.65% (66/69), the negative prediction value was 85.71% (48/56), the total consistency rate was 91.20% (114/125), and the Kappa value was 0.82. Conclusion: A rapid detection system of MTBC and NTM based on CPA technology was successfully established, which could provide a new detection method for the prevention and control of tuberculosis epidemic in primary hospitals.

Key words: Nucleic acid amplification techniques, Mycobacterium tuberculosis, Mycobacterium, atypical

CLC Number:

R378.91

Huang Wenbin, Chen Liping, Zhang Wang, Xiao Ting, Zhang Mane, Wu Dingchang. Establishment of a cross primer isothermal amplification technique for rapid detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria[J]. Chinese Journal of Antituberculosis, 2023, 45(8): 734-743. doi: 10.19982/j.issn.1000-6621.20230088

Figures/Tables 11

References 21

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组名	引物名称	引物和探针序列
MTBC/NTM-16S-1	BF-1	5'-TGCAGGGGAGACTGGAAT-3'
	BR-1	5'-GTACTCCCCAGGCGGGG-3'
	CPF-1	5'-GCGTCAGTTACTGCCCAGAGAC-TGGAATGCGCAGATATCAGG-3'
	CPR-1	5'-CTGGTAGTCCACGCCGTAAACG-GTTAGCTACGGCACGGATC-3'
	LF-1	5'-CGCCTTCGCCACCGGTGT-3'
	LR-1	5'-TGTGGGTTTCCTTCCTTG-3'
MTBC/NTM-16S-2	BF-2	5'-GGAATTCCTGGTGTAGCGG-3'
	BR-2	5'-CCAGGCGGGGTACTTAATG-3'
	CPF-2	5'-CAGCGTCAGTTACTGCCCAGAG-TGCGCAGATATCAGGAGGA-3'
	CPR-2	5'-GTAGTCCACGCCGTAAACGGT-GTTAGCTACGGCACGGATC-3'
	LF-2	5'-CGCCTTCGCCACCGGTGT-3'
	LR-2	5'-TGTGGGTTTCCTTCCTTG-3'
MTBC/NTM-16S-3	BF-3	5'-GGGAGACTGGAATTCCTGG-3'
	BR-3	5'-CCAGGCGGGGTACTTAATG-3'
	CPF-3	5'-CAGCGTCAGTTACTGCCCAGA-GCGCAGATATCAGGAGGAAC-3'
	CPR-3	5'-CTGGTAGTCCACGCCGTAAACG-GTTAGCTACGGCACGGATC-3'
	LF-3	5'-AGACCCGCCTTCGCC-3'
	LR-3	5'-GTGGGTACTAGGTGTGGGTT-3'
MTBC/NTM-16S-4	BF-4	5'-GGAATTCCTGGTGTAGCGG-3'
	BR-4	5'-CAGGCGGGGTACTTAATGC-3'
	CPF-4	5'-CAGCGTCAGTTACTGCCCAGAG-TGCGCAGATATCAGGAGGA-3'
	CPR-4	5'-CTGGTAGTCCACGCCGTAAACG-GTTAGCTACGGCACGGATC-3'
	LF-4	5'-AGACCCGCCTTCGCC-3'
	LR-4	5'-GTGGGTACTAGGTGTGGGTT-3'
MTBC/NTM-16S-5	BF-5	5'-GAGTACTGCAGGGGAGACT-3'
	BR-5	5'-GGATCCCAAGGAAGGAAACC-3'
	CPF-5	5'-GCGTCAGTTACTGCCCAGAGAC-AATGCGCAGATATCAGGAGG-3'
	CPR-5	5'-CTGAGGAGCGAAAGCGTGGG-ACACCTAGTACCCACCGTT-3'
	LF-5		5'-CCGGTGTTCCTCCTGATATC-3'
	LR-5		5'-ACCCTGGTAGTCCACGCC-3'
MTBC/NTM-16S-6	BF-6		5'-GGAATTCCTGGTGTAGCGG-3'
	BR-6		5'-CAGGCGGGGTACTTAATGC-3'
	CPF-6		5'-GCGTCAGTTACTGCCCAGAGAC-AATGCGCAGATATCAGGAGG-3'
	CPR-6		5'-CTGGTAGTCCACGCCGTAAACG-GTTAGCTACGGCACGGATC-3'
	LF-6		5'-CGCCTTCGCCACCGGTGT-3'
	LR-6		5'-GTGGGTACTAGGTGTGGGTT-3'
MTBC/NTM-16S-探针	FAM-1		5'-6-FAM-CCGAGCGATTCGCTCGG-(Int BHQ1 dT)-TGTGGGTTTCCTTCCTTG-3'
	FAM-2		5'-6-FAM-CCGAGCGATTCGCTCGG-(Int BHQ1 dT)-ACGGTGGGTACTAGGTGTGGGTTTCC-3'
	FAM-3		5'-6-FAM-CCGAGCGATTCGCTCGG-(Int BHQ1 dT)-GTGGGTACTAGGTGTGGGTT-3'

反应体系成分	初始浓度	终浓度	加样量(μl/人份)
BF引物	100μmol/L	0.1μmol/L	0.02
BR引物	100μmol/L	0.1μmol/L	0.02
CPF引物	100μmol/L	0.9μmol/L	0.18
CPR引物	100μmol/L	0.9μmol/L	0.18
LF引物	100μmol/L	0.7μmol/L	0.14
LR引物	100μmol/L	0.1μmol/L	0.02
BstDNA聚合酶	8U/μl	0.32U/μl	1.00
2×预混缓冲液^a	-	-	12.50
SYTO9	-	-	0.40
质粒模板	-	-	5.00
双蒸水	-	-	5.54
总体积	-	-	25.00

反应体系成分	初始浓度	终浓度	加样量(μl/人份)
BF引物	100μmol/L	0.1μmol/L	0.02
BR引物	100μmol/L	0.1μmol/L	0.02
CPF引物	100μmol/L	0.9μmol/L	0.18
CPR引物	100μmol/L	0.9μmol/L	0.18
LF引物	100μmol/L	0.7μmol/L	0.14
不同探针	100μmol/L	0.1μmol/L	0.02
BstDNA聚合酶	8U/μl	0.32U/μl	1.00
2×预混缓冲液^a	-	-	12.50
质粒模板	-	-	5.00
双蒸水	-	-	5.94
总体积	-	-	25.00

引物名称	引物探针序列
MTBC/NTM-16S-BF-6	5'-GGAATTCCTGGTGTAGCGG-3'
MTBC/NTM-16S-BR-6	5'-CAGGCGGGGTACTTAATGC-3'
MTBC/NTM-16S-CPF-6	5'-GCGTCAGTTACTGCCCAGAGAC-AATGCGCAGATATCAGGAGG-3'
MTBC/NTM-16S-CPR-6	5'-CTGGTAGTCCACGCCGTAAACG-GTTAGCTACGGCACGGATC-3'
MTBC/NTM-16S-LF-6	5'-CGCCTTCGCCACCGGTGT-3'
MTBC/NTM-16S-LR-FAM-3	5'-6-FAM-CCGAGCGATTCGCTCGG-(Int BHQ1 dT)-GTGGG TACTAGGTGTGGGTT-3'

16S rRNA质粒浓度	平均Ct值	检测结果	16S rRNA质粒浓度	平均Ct值	检测结果
1×10⁷拷贝/μl	5.40	阳性	500拷贝/μl	9.85	阳性
1×10⁶拷贝/μl	6.27	阳性	200拷贝/μl	10.87	阳性
1×10⁵拷贝/μl	7.17	阳性	100拷贝/μl	12.27	阳性
1×10⁴拷贝/μl	8.13	阳性	50拷贝/μl	>40	阴性
1×10³拷贝/μl	9.45	阳性	双蒸水	>40	阴性