Loop-Mediated Isothermal Amplification for Rapid and Reliable Diagnosis of Tuberculous Meningitis

Abstract

Diagnosis of tuberculous meningitis (TBM) is often difficult.A reliable, simple, and rapid diagnostic test that can be performed in any standard laboratory could be helpful in TBM diagnosis. In this study, a loop-mediated isothermal amplification assay (LAMP) was evaluated to rapidly detect and diagnose TBM infection and was compared to the performance of nested PCR. Six specific primers were used to recognize the IS6110 genomic sequence from Mycobacterium tuberculosis, which included one forward outer primer, one reverse outer primer, two respective inner primers, and two loop primers. The optimum reaction temperature and time were 63°C and 60 min, respectively. Nested PCR was performed targeting the IS6110 region from M. tuberculosis using a commercial kit. The LAMP method yielded a sensitivity of 88.23% and a specificity of 80%, compared to the nested-PCR assay, which yielded a sensitivity of 52.9% and a specificity of 90% for TBM diagnosis. Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect TBM infection and that it is superior to the nested-PCR assay. LAMP is very simple, and it can be performed in any laboratory and in rural settings.

Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic resistance in tuberculosis

“…The polymerase chain reaction (PCR) is the most common methodology utilised in NAAT; alternatives include real-time PCR, isothermal strand displacement amplification or transcription-mediated amplification, and the ligase chain reaction. [27][28][29][30] Although NAAT can theoretically detect a single copy of nucleic acid in a specimen, sensitivity can be significantly compromised by the presence of PCR inhibitors in clinical specimens and loss of nucleic acids during specimen processing, and therefore tends to vary.…”

Loop-mediated isothermal amplification for rapid molecular detection of Enterocytozoon bieneusi in faecal specimens

“…Even though the sensitivity and specificity of the microsporidial LAMP were high, several false-positive and false-negative results were also obtained using this assay. Thus, analysis using sequencing and restriction enzymes would provide a great help in distinguishing between these results and contamination (Nagdev et al, 2011). Therefore, further analysis between these LAMP-negative but PCR-positive samples was done by sequencing.…”

“…The samples that were LAMPpositive but negative by PCR may be due to the presence of inhibitors in the samples, which are known to have a stronger inhibitory effect on the polymerases used in PCR than on the Bst enzyme used in LAMP. Nonetheless, further study needs to be undertaken by incorporating internal amplification controls to determine whether there is any inhibition occurring in the PCR and LAMP assays that may be responsible for these results (Nagdev et al, 2011). Using microscopy, researchers can detect the spores but not the species.…”

“…The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated as described by Nagdev et al (2011) using PCR as the reference test.…”

Validating the Loop Mediated Isothermal Amplification (LAMP) technique to detect tuberculosis in a Sri Lankan laboratory setting

“…The LAMP reaction was carried out using Mycobacterium tuberculosis specific primers targeting the rimM gene sequence as previously described, with few modifications as follows; a. Varying amounts of SYBR dye; 1μl of 1/10 diluted dye, 5μl of 1/10 diluted dye and 1μl of undiluted dye, was added, b. Betaine at a concentration of 0.8M was also included in the reaction mixture, c. The ZYBR green dye was placed on the inner-side of the cap of the microfuge tube containing the reaction mixture prior to amplification, and then the tube was spun after the amplification to mix the dye with the LAMP amplified products [4,6].…”