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BRIEF COMMUNICATION
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 302-304
 

Development and evaluation of a loop-mediated isothermal amplification combined with au-nanoprobe assay for rapid detection of Mycobacterium tuberculosis


1 Innovative Learning Center, Srinakharinwirot University, Bangkok 10110, Thailand
2 Department of Clinical Pathology, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, Bangkok 10110, Thailand
3 Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand
4 Department of Pathology, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand

Date of Web Publication5-Jul-2017

Correspondence Address:
Thongchai Kaewphinit
Innovative Learning Center, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_15_333

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 ~ Abstract 

Loop-mediated isothermal amplification (LAMP) has been proposed as an inexpensive and easy to perform assay for molecular diagnostics. We present a novel strategy for the detection of LAMP amplicons derived from Mycobacterium tuberculosis by the use of Au-nanoprobes. When applied to a total of 93 clinical specimens, the LAMP assay demonstrated sensitivity and specificity higher than that of polymerase chain reaction and culture. The Au-nanoprobe augmented LAMP test platform with its advantages of robust reagents and a simple colorimetric detection method can be adapted easily for the rapid detection of other infectious disease agents at a low cost.


Keywords: Au-nanoprobe, diagnosis, loop-mediated isothermal amplification, Mycobacterium tuberculosis, tuberculosis


How to cite this article:
Kaewphinit T, Ckumdee J, Chansiri K, Santiwatanakul S. Development and evaluation of a loop-mediated isothermal amplification combined with au-nanoprobe assay for rapid detection of Mycobacterium tuberculosis. Indian J Med Microbiol 2017;35:302-4

How to cite this URL:
Kaewphinit T, Ckumdee J, Chansiri K, Santiwatanakul S. Development and evaluation of a loop-mediated isothermal amplification combined with au-nanoprobe assay for rapid detection of Mycobacterium tuberculosis. Indian J Med Microbiol [serial online] 2017 [cited 2017 Sep 26];35:302-4. Available from: http://www.ijmm.org/text.asp?2017/35/2/302/209565



 ~ Introduction Top


The diagnosis of tuberculosis (TB) in the laboratory still depends on microscopy or culture. Microscopy, despite being inexpensive and fast, has low sensitivity. Therefore, culture is the reference test for diagnostic confirmation; however, it takes four to 8 weeks to reach a conclusion in negative specimens.

Loop-mediated isothermal amplification (LAMP) has been proposed as an inexpensive and easy to perform assay for molecular diagnostics and has been developed as an alternative method to polymerase chain reaction (PCR). It is capable of amplifying target DNA under isothermal condition within <1 h.[1] However, LAMP-Mycobacterium tuberculosis (MTB) amplicons may also be assessed indirectly using a lateral flow dipstick assay [2] or using an Au-nanoparticle (AuNP) probe assay.[3] The latter is at a preliminary stage of study and has never been evaluated in field samples.

In the present study, we evaluated LAMP combined with an AuNP probe assay for the rapid direct detection of MTB and compared it to the smear and culture assay in clinical field samples.


 ~ Materials and Methods Top


This study was reviewed and approved by the Expedited Review Ethics Committee of the University. All clinical samples (93 sputum samples) and standard strains (H37Rv) were provided by the National Tuberculosis Reference Laboratory, Bureau of Tuberculosis, Department of Disease Control, Ministry of Public Health, Thailand.

LAMP reaction and the detection of LAMP amplicons were performed by a modification of a previous method.[3] Sensitivity and specificity were examined using 10-fold serial dilutions prepared from colonies of H37Rv, and the specificity of LAMP primers was examined using DNA extracted from other mycobacteria. Ninety-three clinical sputum samples were tested individually with the LAMP-AuNP probe assay; the results were compared to those of the standard culture assay and acid-fast bacilli (AFB) microscopy.

Specimens were divided into the following groups: (a) smear positive and culture positive (S+C+) (n = 59) and (b) smear negative but culture positive (S−C+) (n = 14). Other microorganisms were taken as negative controls (n = 20).


 ~ Results Top


Synthesised AuNP probe had one absorption peak at ~525 nm, while the LAMP-AuNP probe absorbed maximally at ~530 nm [Figure 1]. The effect of salt concentration on aggregation was tested by a final concentration of 0.5 M MgSO4 which gave the best discrimination between positive and negative results [Figure 2]. With the 5 nM AuNP probe, the best colour intensity was seen at ratios between 8:2 and 5:5, while ratios between 4:6 and 1:9 gave false negative results [Figure 3].
Figure 1: Visible spectra absorption of the Au-nanoparticle oligonucleotide probe, loop-mediated isothermal amplification hybridised with Au-nanoparticle probe (positive), loop-mediated isothermal amplification hybridised with Au-nanoparticle probe (negative) and distilled water with Au-nanoparticle probe (blank) that measured after salt addition

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Figure 2: Effects of ratio of loop-mediated isothermal amplification amplicon to 5-nM Au-nanoparticle probe in tubes contained either were mixed with 5 μl and added of MgSO4 (5 μl fixed volume) at final concentrations 300, 500 and 700 mM

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Figure 3: Optimisation of the Au-nanoparticle hybridisation assay detection of Mycobacterium tuberculosis. Effects of ratio of loop-mediated isothermal amplification amplicon to 5-nM Au-nanoparticle probe in tubes contained either were mixed with 5 μl or added of MgSO4 (5 μl fixed volume) at final concentrations at 500 mM

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When the LAMP-AuNP probe assay was used with 10-fold serial dilutions of DNA (either total DNA extracted from H37Rv target sequence), it was able to detect template at 10 CFU/ml [Figure 4]a; Lanes 1–7]. This result showed identical sensitivity to LAMP followed by AGE [Figure 4]b; Lanes 1–7]. PCR was able to detect template at 100 CFU/ml [Figure 4]c. Therefore, the LAMP-AuNP probe assay was capable of detecting ten time lower concentrations of target DNA than PCR. This LAMP specificity has MTB, whereas none of the control mycobacteria (Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium gordonae) showed a positive result by LAMP-AuNP probe and LAMP-AGE assays, indicating the high specificity of the LAMP-AuNP probe assay [Figure 5].
Figure 4: DNA products amplified by the loop-mediated isothermal amplification-Mycobacterium tuberculosis detected by (a) the LAMP-Au-nanoparticle probe in comparison to (b) LAMP agarose gel electrophoresis and (c) PCR agarose gel electrophoresis using dilutions 107–100 CFU/ml dilutions from Mycobacterium tuberculosis (H37Rv); Lane N: No template as control; Lane M: 100 bp DNA ladder

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Figure 5: Specificity data of the loop-mediated isothermal amplification method for mycobacteria test using 107 CFU/ml of each templates and detection by (a) the loop-mediated isothermal amplification-Au-nanoparticle probe and by (b) gel electrophoresis. Lane M: DNA ladder marker; Lanes 2–7: DNAs of Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium gordonae, respectively. Lane 8: Negative control (no-DNA template)

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The clinical specimens were evaluated using the LAMP-AuNP probe assay; in brief, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the LAMP-AuNP assay for clinical samples were 100% (95% confidence interval [CI], 93.77–100) and 100% (95% CI, 79.95–100), 100% (95% CI, 93.77–100) and 100% (95% CI, 79.95–100), respectively. In contrast, the sensitivity of the AFB smears test was 80.8% (59/73) [Table 1].
Table 1: Clinical samples identified with LAMP-Au-nanoparticle probe tested with specific carrying the IS6110 gene for genotyping, compared with the culture and AFB smear test

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 ~ Discussion Top


Several researchers, including our group, have evaluated LAMP assay for MTB detection. Our LAMP reaction results showed a detection limit at 10 CFU/ml of MTB, indicating that this detection sensitivity was greater than that of PCR. The sensitivity, specificity, PPV and NPV of this assay for the detection of MTB genomic DNA in clinical sample were 100% (95% CI, 93.77–100) and 100% (95% CI, 79.95–100), 100% (95% CI, 93.77–100) and 100% (95% CI, 79.95–100), respectively. A number of previous studies have described LAMP assay targeting the gyrB gene [4] with a detection limit as 5–50 copies, the rrs gene [5] with a detection limit of ten copies and IS6110 with a detection limit of 5 pg of genomic DNA for the detection and diagnosis of MTB infections.[3] Boehme et al.[4] showed that the sensitivity evaluated when targeting the gyrB gene was 97.7% (95%, CI, 95.5–99.9). In addition, Mitarai et al.[6] evaluated LAMP on raw sputum finding that sensitivity was 98.2% (95% CI 94.9–99.4) while the sensitivity in smear-negative, culture-positive specimens was 55.6% (95%, CI 43.4–68.0). The diagnostic sensitivity of direct LAMP for the diagnosis of individuals with TB was 88.2% (95%, CI 81.4–92.7).

This report showed that the combination of LAMP and amplicon detection with AuNP probes could successfully detect MTB-DNA from clinical samples. The LAMP-AuNP probe method showed no cross reaction with other mycobacteria and offered a short total assay time of <1 h. The high sensitivity, relatively short analysis time, low cost and lack of requirement for a thermocycler and separate detection reagents were important advantages of this technique.

Therefore, the LAMP-AuNP MTB probe assay constitutes a safe and simple alternative to traditional PCR for the rapid and sensitive detection of MTB DNA.

Acknowledgment

This work was supported by Srinakharinwirot University. The researchers would like to acknowledge the help of the Bureau of Tuberculosis, Ministry of Public Health, Thailand. We would also like to thank Dr. Kamolwan Karomprach Klaykaew who assisted with the statistical analysis of the results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:E63.  Back to cited text no. 1
[PUBMED]    
2.
Kaewphinit T, Arunrut N, Kiatpathomchai W, Santiwatanakul S, Jaratsing P, Chansiri K. Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int 2013;2013:926230.  Back to cited text no. 2
[PUBMED]    
3.
Kaewphinit T, Santiwatanakul S, Chansiri K. Colourimetric DNA based biosensor combined with loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis by using gold nanoprobe aggregation. Sens Transducers 2013;149:123-8.  Back to cited text no. 3
    
4.
Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M. Operational feasibility of using loop mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy center of developing countries. J Clin Microbiol 2007;45:1936-40.  Back to cited text no. 4
    
5.
Pandey BD, Poudel A, Yoda T, Tamaru A, Oda N, Fukushima Y, et al. Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients. J Med Microbiol 2008;57(Pt 4):439-43.  Back to cited text no. 5
    
6.
Mitarai S, Okumura M, Toyota E, Yoshiyama T, Aono A, Sejimo A, et al. Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis. Int J Tuberc Lung Dis 2011;15:1211-7, i.  Back to cited text no. 6
[PUBMED]    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]



 

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