|Year : 2015 | Volume
| Issue : 4 | Page : 568-571
Loop mediated isothermal amplification assay using hydroxy naphthol blue, conventional polymerase chain reaction and real-time PCR in the diagnosis of intraocular tuberculosis
PK Balne1, S Basu2, S Rath2, MR Barik1, S Sharma3
1 Ocular Microbiology Service, L V Prasad Eye Institute, Bhubaneswar, Odisha, India
2 Retina-Vitreous and Uveitis Services, L V Prasad Eye Institute, Bhubaneswar, Odisha, India
3 Jhaveri Microbiology Centre, L V Prasad Eye Institute, Hyderabad, Telangana, India
|Date of Submission||28-Oct-2014|
|Date of Acceptance||23-Apr-2015|
|Date of Web Publication||16-Oct-2015|
Jhaveri Microbiology Centre, L V Prasad Eye Institute, Hyderabad, Telangana
Source of Support: Department of Science and Technology,
Government of India, New Delhi, India, Grant no. SR/FT/LS-15/2011,
awarded to Soumyava Basu and Hyderabad Eye Research
Foundation, Hyderabad. Praveen Kumar Balne is supported by
a senior research fellowship from the Council of Scientific and
Industrial Research (CSIR), Government of India, New Delhi, India, Conflict of Interest: None
This study is a comparative evaluation (Chi-square test) of a closed tube loop mediated isothermal amplification assay using hydroxy naphthol blue dye (HNB-LAMP), real-time polymerase chain reaction (PCR) and conventional PCR in the diagnosis of intraocular tuberculosis. Considering clinical presentation as the gold standard in 33 patients, the sensitivity of HNB-LAMP assay (75.8%) was higher (not significant, P value 0.2) than conventional PCR (57.6%) and lower than real-time PCR (90.9%). Specificity was 100% by all three methods. No amplification was observed in negative controls (n = 20) by all three methods. The cost of the HNB-LAMP assay was Rs. 500.00 and it does not require thermocycler, therefore, it can be used as an alternative to conventional PCR in resource-poor settings.
Keywords: Hydroxynaphthol blue, loop mediated isothermal amplification assay, MPB64, ocular tuberculosis, real-time polymerase chain reaction
|How to cite this article:|
Balne P K, Basu S, Rath S, Barik M R, Sharma S. 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;33:568-71
|How to cite this URL:|
Balne P K, Basu S, Rath S, Barik M R, Sharma S. 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 [serial online] 2015 [cited 2019 Jun 26];33:568-71. Available from: http://www.ijmm.org/text.asp?2015/33/4/568/167339
| ~ Introduction|| |
Intraocular tuberculosis is a paucibacillary disease, which is difficult to diagnose by conventional methods. Wroblewski et al., reported that only 1 or 2 Mycobacterium tuberculosis bacteria were found in the vicinity of giant cells or areas of necrosis in 42 cases of histopathologically proven ocular tuberculosis over a period of 75 year. Polymerase chain reaction (PCR) has emerged as a promising tool for definitive diagnosis of this condition, but requires high-cost precision instruments. Loop mediated isothermal amplification (LAMP) assay amplifies the DNA under isothermal conditions (60–65°C) with high degrees of specificity, sensitivity, efficiency, and rapidity. LAMP assay has been developed for numerous human and animal pathogens. We reported a LAMP assay in 2013 for the diagnosis of intraocular tuberculosis targeting the gene MPB64, wherein SYBR green dye was used post-amplification for visualisation of the reaction. Similar assay was reported recently for the diagnosis of tubercular uveitis using primers for IS6110 gene. We noted occasional incidents of amplicon contamination (false positive results in negative controls) in the laboratory, which made us seek safer methods. We believe that post-amplification opening of the tube to add SYBR green was the potential source of contamination as it may cause amplicon aerosol contamination of the surroundings. This prompted us to develop an alternative protocol for LAMP assay that uses hydroxy naphthol blue dye (HNB-LAMP assay) in the tubes prior to the amplification. This study reports the utility of HNB-LAMP assay for the diagnosis of intraocular tuberculosis in comparison with conventional PCR and TaqMan based real-time PCR.
| ~ Materials and Methods|| |
The study was approved by the local institutional review board and informed consent was taken from all the patients. Thirty-three intraocular samples (16 vitreous and 17 aqueous humour) from patients with clinically suspected intraocular tuberculosis based on criteria described earlier  and 20 aqueous humour samples from patients undergoing cataract surgery (negative controls), were included in the study. Up to 200 µL of the ocular sample was aseptically collected from each patient and stored at −20°C till processed. All samples were processed within a week after collection. After DNA extraction using QIAamp DNA mini kit (Qiagen, Germany) all samples were tested for M. tuberculosis DNA by HNB-LAMP assay, conventional PCR and real-time PCR targeting MPB64 gene in M. tuberculosis H37Rv strain (Genbank accession number NC_018143). Same DNA extraction protocol was used for extraction of DNA from M. tuberculosis culture and used as a positive control for all the assays in the study. With a small volume of clinical sample and an extremely low load of organisms, it was not possible to spare DNA for quantification prior to testing. The extracted DNA was not quantified and directly used for the HNB-LAMP assay, conventional PCR, and real-time PCR.
Hydroxy naphthol blue-LAMP assay was standardised according to a previously published protocol  with 2 modifications, 1 µL of 150 mM MgSO4, 1 µL of 3 mM hydroxy naphthol blue dye (Sigma-Aldrich, St. Louis, MO, USA) added prior to amplification. After amplification, the reaction tubes were placed on a light box and a naked eye observation of colour change from violet to blue indicated positive reaction [Figure 1]a. LAMP products were also verified by 2% agarose gel electrophoresis with ultraviolet light transillumination which showed ladder like banding pattern [Figure 1]b. Conventional PCR was carried out as described previously. For real-time PCR Custom TaqMan ® Gene Expression Assay (Applied Biosystems, Foster City, California, USA) consisting of a set of two primers [MTB_F (5'- GTTCTGATAATTCACCGGGTCCAA- 3') and MTB_R (5'-AGACCGGACAACAGGTATCGA -3')] and a FAM™ dye-labeled TaqMan ® MGB probe (5'-TAGCGCCGAATGCC-3') targeting MPB64 gene in M. tuberculosis H37Rv strain were used in accordance with the manufacturer's instructions. All procedures like DNA extraction, master mixture preparation, addition of template, amplification, and gel electrophoresis were done in separate rooms and dedicated laminar flow hoods and pipettes were used for each step to avoid contamination.
|Figure 1: Results of four patients samples with presumed intraocular tuberculosis,,, tested for Mycobacterium tuberculosis DNA by three methods. (a) Hydroxy naphthol blue-loop mediated isothermal amplification (HNB-LAMP) assay: Positive reaction indicated by blue colour is seen in tubes 1–4 and positive control (PC), while a violet colour indicated a negative reaction in tube negative control (NC). (b) Agarose gel electrophoresis (2%) picture of HNB-LAMP assay showing ladder like banding pattern in lane 1–4 and PC. (c) Agarose gel electrophoresis (1.5%) picture of uniplex polymerase chain reaction (PCR) showing 240 base pairs PCR amplified products in lane 1, 3 and 4 (patient samples) and PC1 and PC2. No amplification in lane 2 (patient sample which was positive by HNB-LAMP and real time PCR) and NC. MW: 100 base pair molecular weight DNA ladder (d) Real-time PCR amplification plot showing amplification in four patient samples (inside the circle) and standards 5 × 108–5 × 104 copies (without circle from left to right)|
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Different concentrations of M. tuberculosis H37Rv DNA was used to test the analytical sensitivity of the three methods. Analytical specificity was tested using DNA from different microorganisms (bacteria, fungi, virus and Acanthamoeba sp. ) and human leucocytes DNA.
| ~ Results|| |
The analytical sensitivity of HNB-LAMP assay and real-time PCR was 10 fg/reaction (2 copies/reaction) and for conventional PCR 20 fg/reaction (4 copies/reaction). In HNB-LAMP, conventional PCR and real-time PCR assays, positive reaction was detected only with M. tuberculosis DNA and not with any other DNA tested for specificity. Of the 33 patient samples from suspected cases of intraocular tuberculosis, 25 (75.8%) were positive by HNB-LAMP assay, 19 (57.6%) by conventional PCR and 30 (90.91%) by real-time PCR. [Figure 1]c shows the agarose gel electrophoresis picture of conventional PCR and [Figure 1]d shows the real-time PCR amplification plot in 4 patient samples and standards. No amplification was observed in negative control samples by all three methods. Considering clinical presentation as the gold standard, the sensitivity, specificity, and predictive values of HNB-LAMP, conventional PCR and real-time PCR [Table 1] were calculated by MedCalc statistical software (http://www.medcalc.org). The sensitivity of HNB-LAMP assay (75.8%) was higher than conventional PCR (57.6%) and lower than real-time PCR (90.9%)), but the differences were statistically (Chi-square test) not significant (P value 0.2).
|Table 1: Sensitivity, specificity, and predictive values of HNB-LAMP assay, conventional PCR and real-time PCR for the diagnosis of intraocular tuberculosis|
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| ~ Discussion|| |
The LAMP assay is an isothermal amplification method and the amplification products can be detected by visual reading with the naked eye in normal room light. Detection of turbidity by the naked eye is the simplest and most cost-effective method for judging a positive LAMP reaction. But this method requires some skill for assessing the result. Different DNA intercalating dyes such as SYBR green, picogreen, and propidium iodide have been used for better visibility of the LAMP reaction result.,,, These dyes are added to the LAMP reaction after the amplification is completed. Opening the reaction tube to add these dyes may result in amplicon aerosol contamination of the surroundings. To overcome this, metal ion indicators, such as calcein and HNB dye have been successfully used to visualise the LAMP reaction result.,, These metal ion indicators can be added to the pre-reaction solution and the positive reaction is indicated by colour change. Goto et al., compared the LAMP assay using HNB, SYBR green, and calcein dyes and reported that the detection limit of HNB-LAMP assay was equivalent to that of the assay using SYBR green dye. In contrast, the detection limit of the LAMP assay using calcein was 10 times lower than those of other assays. In our study, HNB-LAMP detection limit was found to be 2 copies/reaction, which was equal to our previously reported LAMP assay using SYBR green dye.
We chose MPB64 gene as the target for detection of M. tunberculosis complex DNA since significant number of clinical isolates of M. tuberculosis in India may have low (1–2) or no copy of the IS6110 gene element. This is corroborated by the recently described LAMP assay using this gene target, wherein 3 out of 10 cases were missed. The authors found MPB64 in these 3 missed cases in the multitarget PCR. Although the gold standards were different (Clinical versus multitarget PCR) the sensitivity of HNB-LAMP assay in our study with MPB64 (75.8%) was higher than recently reported LAMP assay with IS6110 gene target (70%).
Advantages of LAMP assay include its limited requirement for space and instrumentation which makes it suitable for small scale diagnostic laboratories. HNB-LAMP assay is a closed-tube method which prevents the risk of cross-contamination and the colour change in the reaction can be clearly appreciated by the naked eye. It does not require thermal cycler, gel electrophoresis unit and gel documentation system, and laminar flow hoods. An analysis of the cost of reagents, man power and power consumption showed that one HNB-LAMP test, conventional PCR and real time PCR would cost Rs. 500.00 (10 US dollars), Rs. 1000.00, and Rs. 3000.00 respectively. Though the detection limit of HNB-LAMP assay and real-time PCR was same, the negative predictive value and the sensitivity of the HNB-LAMP was less than real time PCR. It might be due to the presence of unknown DNA amplification inhibitors in the ocular samples or repeated freeze-thaw of the DNA for different assays.
The time taken for DNA extraction and identification was around 7 h for conventional PCR and 4 h for HNB-LAMP and real-time PCR. Based on these results, we suggest adaptation of HNB-LAMP assay as an alternative diagnostic tool to conventional PCR for rapid diagnosis of intraocular tuberculosis in resource-poor settings. We believe that this assay has a potential to be used, and should be evaluated for its efficacy, in the diagnosis of other extrapulmonary paucibacillary tuberculosis.
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