A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

Abstract

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

A Case of Bacillus Calmette-Guérin Cystitis Diagnosed with a Novel Loop-mediated Isothermal Amplification Method

“…Nucleic acid amplification by the newly developed LAMP method needs only a heating block to amplify the specific target gene and takes only 60 minutes to complete 3 . In the present case it detected BCG as accurately as PCR did and was thus demonstrated to be useful for detecting BCG in clinical settings faster and more easily.…”

“…This method uses an oligonucleotide LAMP primer designed to detect the lack of the RD1 sequence and amplifies the BCG gene specifically. 3 …”

“…We report herein the first case of BCG cystitis diagnosed with a newly developed easy-to-use LAMP method 3 designed to detect the lack of RD1, that is, to amplify the BCG gene specifically.…”

Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis

“…In comparison to previous reports, a LAMP system targeting RD1 deletions for identification of M . bovis BCG required more than 200 copies of the targets for detection using a turbidimeter, and 2000 copies for detection with a visible color change [ 42 ]. In other reports, a LAMP system targeting the rim -encoding 16S rRNA-processing protein for detection of M .…”

Rapid and sensitive detection of Mycobacterium tuberculosis based on strand displacement amplification and magnetic beads

“…For example, disappointing detection rates, poor sensitivity, and low throughput of smear microscopy methods [ 6 ] ; insufficient specificity of chest radiography for immune‐compromised and HIV‐infected patients [ 7 ] ; the requirement for sophisticated laboratory equipment and the slow growth of Mycobacterium tuberculosis (MTB) bacteria have limited the wide application of culture methods. [ 8 ] In recent years, nucleic acid amplification technologies such as polymerase chain reaction (PCR), [ 9–11 ] rolling circle amplification (RCA) [ 12–14 ] and loop‐mediated isothermal amplification, [ 15,16 ] have been applied in TB diagnosis for high sensitivity and rapid analysis. As these techniques require enzymes for signal amplification and need rigorous controls to ensure validity and accuracy, the wide application of these methods has been limited by the cost of tests, and the need for well trained personnel and sophisticated experimental conditions.…”