Visualized detection for <i>mycobacterium</i> tuberculosis using loop-mediated isothermal amplification assay_Journal of Biomedical Engineering

Authors：

ZHAO Na ^1,2 , SUN Dianxing ² ,  LIU Jinxia ¹

1. The Experiment Center of Pathogenic Biology, Chengde Medical University, Chengde, Hebei 067000, P.R.China;
2. The Center of Diagnosis and Treatment of Liver Disease, Bethune International Peace Hospital, Shijiazhuang 050082, P.R.China;

Corresponding author：

LIU Jinxia, Email: 1158210524@qq.com

Keywords：

mycobacterium tuberculosis; loop-mediated isothermal amplification; 16S rDNA; visualized detection

DOI：

10.7507/1001-5515.201606066

Video：

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In this study, loop-mediated isothermal amplification (LAMP) assay in conjunction with calcein for visualized detection of Mycobacterium tuberculosis (MTB) was established. Firstly, four LAMP primers were designed according to the region of 16S rDNA sequences of MTB. Secondly, clinical sputum samples were collected, decontaminated and their DNA was extracted. Thirdly, standard MTB strains were used to evaluate the specificity and sensitivity of LAMP. At the same time, electrophoresis was used for products detection and calcein was used for visualized verification. At last, Chi-squared test function in SPSS 17.0 software was used for consistency evaluation of LAMP assay as compared with the gold standard (culture method). Results showed that there was no nonspecific amplification appeared in the specificity assay and the detection limit was 10 copies/tube in the sensitivity assay. In addition, visualized method by calcein had a comparable sensitivity with that of electrophoresis method. After evaluation of clinical practicability, the sensitivity of LAMP was calculated as 94.74% and the specificity was 90%, respectively. And Chi-squared test showed that LAMP and culture method had no statistic difference, and the two methods were in good consistency (P>0.05). In conclusion, LAMP assay introduced in our study has the characteristics of high efficiency and visualized detection so that this technique has great application prospects in the resource-limited environment, such as work field and primary care hospitals.

Citation： ZHAONa, SUNDianxing, LIUJinxia. Visualized detection for mycobacterium tuberculosis using loop-mediated isothermal amplification assay. Journal of Biomedical Engineering, 2017, 34(1): 72-77. doi: 10.7507/1001-5515.201606066 Copy

1.	Kaewphinit T, Arunrut N, Kiatpathomchai W, et al. Detection of mycobacterium tuberculosis by using Loop-Mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int, 2013: 1-6.
2.	Creecy A, Russ P K, Solinas F, et al. Tuberculosis biomarker extraction and isothermal amplification in an integrated diagnostic device. PLoS One, 2015, 10(7): e0130260.
3.	Bai Yuanyuan, Wang Yueling, Shao Chunhong, et al. GenoType MTBDRplus assay for rapid detection of multidrug resistance in mycobacterium tuberculosis: a Meta-Analysis. PLoS One, 2016, 11(3): e0150321.
4.	Buss B F, Keyser-Metobo A, Rother J, et al. Possible airborne Person-to-Person transmission of mycobacterium bovis - Nebraska 2014-2015. MMWR Morb Mortal Wkly Rep, 2016, 65(8): 197-201.
5.	周东鹏, 李慧娟, 沙丽. 378 株结核分枝杆菌耐药状况的临床分析. 中国实用医刊, 2011, 38(22): 111-112.
6.	Manjelievskaia J, Erck D, Piracha S, et al. Drug-resistant TB: deadly, costly and in need of a vaccine. Trans R Soc Trop Med Hyg, 2016, 110(3, SI): 186-191.
7.	Li Yan’An, Cao Xinrui, Li Shiming, et al. Characterization of mycobacterium tuberculosis isolates from Hebei, China: genotypes and drug susceptibility phenotypes. BMC Infect Dis, 2016, 16(1): 107.
8.	Li Furong, Gao Bo, Xu Wei, et al. The defect in autophagy induction by clinical isolates of mycobacterium tuberculosis is correlated with poor tuberculosis outcomes. PLoS One, 2016, 11(1): e0147810.
9.	Liaquat A, Iram S, Hussain S, et al. Concomitant presence of culture-proven active pulmonary tuberculosis in patients with chronic obstructive pulmonary disease - A hospital based study. Pak J Med Sci, 2015, 31(6): 1344-1348.
10.	Demers A M, Venter A, Friedrich S O, et al. Direct susceptibility testing of mycobacterium tuberculosis for pyrazinamide by use of the bactec MGIT 960 system. J Clin Microbiol, 2016, 54(5): 1276-1281.
11.	Choi W H. Evaluation of anti-tubercular activity of linolenic acid and conjugated-linoleic acid as effective inhibitors against Mycobacterium tuberculosis. Asian Pac J Trop Med, 2016, 9(2): 121-125.
12.	Tans-Kersten J, Lin Shou Y G, Desmond E A. Evaluating shared laboratory services: detecting mycobacterium tuberculosis complex and drug resistance using molecular and Culture-Based methods. Public Health Rep, 2016, 131(1): 117-125.
13.	Choi Y, Hong S R, Jeon B Y, et al. Conventional and real-time PCR targeting 165 ribosomal RNA for the detection of Mycobacterium tuberculosis complex. Int J Tuberc Lung Dis, 2015, 19(9): 1102-1108.
14.	Padya L, Chinombe N, Magwenzi M, et al. Molecular identification of mycobacterium species of public health importance in cattle in Zimbabwe by 16S rRNA gene sequencing. Open Microbiol J, 2015, 9: 38-42.
15.	姜广路, 黄海荣, 赵雁林, 等. 痰涂片阳性结核病患者的涂片与培养结果分析. 中华结核和呼吸杂志, 2011, 34(5): 353-355.
16.	Ssengooba W, Meehan C J, Lukoye D, et al. Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda. Infect Genet Evol, 2016, 40: 8-16.
17.	Cheon S, Cho H H, Kim J, et al. Recent tuberculosis diagnosis toward the end TB strategy. J Microbiol Methods, 2016, 123: 51-61.
18.	Mutingwende I, Vermeulen U, Steyn F, et al. Development and evaluation of a rapid multiplex-PCR based system for Mycobacterium tuberculosis diagnosis using sputum samples. J Microbiol Methods, 2015, 116: 37-43.
19.	Cross L J, Anscombe C, Mchugh T D, et al. A rapid and sensitive diagnostic screening assay for detection of mycobacteria including mycobacterium tuberculosis directly from sputum without extraction. Int J Bacteriol, 2015: 593745.
20.	Cezar R D, Lucena-Silva N, Filho A F, et al. Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil. BMC Vet Res, 2016, 12(1): 31.
21.	Balne P K, Basu S, Rath S, et al. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis. Indian J Med Microbiol, 2015, 33(4): 568-571.
22.	Neonakis I K, Spandidos D A, Petinaki E. Use of loop-mediated isothermal amplification of DNA for the rapid detection of Mycobacterium tuberculosis in clinical specimens. Eur J Clin Microbiol Infect Dis, 2011, 30(8): 937-942.
23.	INOSHIMA Y, ISHIGURO N. Evaluating the stability of loop-mediated isothermal amplification reagents at irregular storage temperatures for On-Site diagnosis. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 2015, 26(1): 7-12.
24.	Yang Q, Wang F, Prinyawiwatkul W, et al. Robustness of salmonella loop-mediated isothermal amplification assays for food applications. J Appl Microbiol, 2014, 116(1): 81-88.
25.	Melville L, Kenyon F, Javed S, et al. Development of a loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Haemonchus contortus eggs in ovine faecal samples. Vet Parasitol, 2014, 206(3/4): 308-312.
26.	Cloud J L, Neal H, Rosenberry R, et al. Identification of mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J Clin Microbiol, 2002, 40(2): 400-406.

1. Kaewphinit T, Arunrut N, Kiatpathomchai W, et al. Detection of mycobacterium tuberculosis by using Loop-Mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int, 2013: 1-6.
2. Creecy A, Russ P K, Solinas F, et al. Tuberculosis biomarker extraction and isothermal amplification in an integrated diagnostic device. PLoS One, 2015, 10(7): e0130260.
3. Bai Yuanyuan, Wang Yueling, Shao Chunhong, et al. GenoType MTBDRplus assay for rapid detection of multidrug resistance in mycobacterium tuberculosis: a Meta-Analysis. PLoS One, 2016, 11(3): e0150321.
4. Buss B F, Keyser-Metobo A, Rother J, et al. Possible airborne Person-to-Person transmission of mycobacterium bovis - Nebraska 2014-2015. MMWR Morb Mortal Wkly Rep, 2016, 65(8): 197-201.
5. 周东鹏, 李慧娟, 沙丽. 378 株结核分枝杆菌耐药状况的临床分析. 中国实用医刊, 2011, 38(22): 111-112.
6. Manjelievskaia J, Erck D, Piracha S, et al. Drug-resistant TB: deadly, costly and in need of a vaccine. Trans R Soc Trop Med Hyg, 2016, 110(3, SI): 186-191.
7. Li Yan’An, Cao Xinrui, Li Shiming, et al. Characterization of mycobacterium tuberculosis isolates from Hebei, China: genotypes and drug susceptibility phenotypes. BMC Infect Dis, 2016, 16(1): 107.
8. Li Furong, Gao Bo, Xu Wei, et al. The defect in autophagy induction by clinical isolates of mycobacterium tuberculosis is correlated with poor tuberculosis outcomes. PLoS One, 2016, 11(1): e0147810.
9. Liaquat A, Iram S, Hussain S, et al. Concomitant presence of culture-proven active pulmonary tuberculosis in patients with chronic obstructive pulmonary disease - A hospital based study. Pak J Med Sci, 2015, 31(6): 1344-1348.
10. Demers A M, Venter A, Friedrich S O, et al. Direct susceptibility testing of mycobacterium tuberculosis for pyrazinamide by use of the bactec MGIT 960 system. J Clin Microbiol, 2016, 54(5): 1276-1281.
11. Choi W H. Evaluation of anti-tubercular activity of linolenic acid and conjugated-linoleic acid as effective inhibitors against Mycobacterium tuberculosis. Asian Pac J Trop Med, 2016, 9(2): 121-125.
12. Tans-Kersten J, Lin Shou Y G, Desmond E A. Evaluating shared laboratory services: detecting mycobacterium tuberculosis complex and drug resistance using molecular and Culture-Based methods. Public Health Rep, 2016, 131(1): 117-125.
13. Choi Y, Hong S R, Jeon B Y, et al. Conventional and real-time PCR targeting 165 ribosomal RNA for the detection of Mycobacterium tuberculosis complex. Int J Tuberc Lung Dis, 2015, 19(9): 1102-1108.
14. Padya L, Chinombe N, Magwenzi M, et al. Molecular identification of mycobacterium species of public health importance in cattle in Zimbabwe by 16S rRNA gene sequencing. Open Microbiol J, 2015, 9: 38-42.
15. 姜广路, 黄海荣, 赵雁林, 等. 痰涂片阳性结核病患者的涂片与培养结果分析. 中华结核和呼吸杂志, 2011, 34(5): 353-355.
16. Ssengooba W, Meehan C J, Lukoye D, et al. Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda. Infect Genet Evol, 2016, 40: 8-16.
17. Cheon S, Cho H H, Kim J, et al. Recent tuberculosis diagnosis toward the end TB strategy. J Microbiol Methods, 2016, 123: 51-61.
18. Mutingwende I, Vermeulen U, Steyn F, et al. Development and evaluation of a rapid multiplex-PCR based system for Mycobacterium tuberculosis diagnosis using sputum samples. J Microbiol Methods, 2015, 116: 37-43.
19. Cross L J, Anscombe C, Mchugh T D, et al. A rapid and sensitive diagnostic screening assay for detection of mycobacteria including mycobacterium tuberculosis directly from sputum without extraction. Int J Bacteriol, 2015: 593745.
20. Cezar R D, Lucena-Silva N, Filho A F, et al. Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil. BMC Vet Res, 2016, 12(1): 31.
21. Balne P K, Basu S, Rath S, et al. Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis. Indian J Med Microbiol, 2015, 33(4): 568-571.
22. Neonakis I K, Spandidos D A, Petinaki E. Use of loop-mediated isothermal amplification of DNA for the rapid detection of Mycobacterium tuberculosis in clinical specimens. Eur J Clin Microbiol Infect Dis, 2011, 30(8): 937-942.
23. INOSHIMA Y, ISHIGURO N. Evaluating the stability of loop-mediated isothermal amplification reagents at irregular storage temperatures for On-Site diagnosis. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 2015, 26(1): 7-12.
24. Yang Q, Wang F, Prinyawiwatkul W, et al. Robustness of salmonella loop-mediated isothermal amplification assays for food applications. J Appl Microbiol, 2014, 116(1): 81-88.
25. Melville L, Kenyon F, Javed S, et al. Development of a loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Haemonchus contortus eggs in ovine faecal samples. Vet Parasitol, 2014, 206(3/4): 308-312.
26. Cloud J L, Neal H, Rosenberry R, et al. Identification of mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J Clin Microbiol, 2002, 40(2): 400-406.

Journal of Biomedical Engineering

Visualized detection for mycobacterium tuberculosis using loop-mediated isothermal amplification assay

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