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

Kouzaki Y, Maeda T, Sasaki H, Tamura S, Hamamoto T, Yuki A, Sato A, Miyahira Y, Kawana A - PLoS ONE (2015)

Bottom Line: 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.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 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.

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 × 10(3) 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 × 10(3) 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.

No MeSH data available.


Related in: MedlinePlus

Alignment of the sequences including RD1, which encodes a 9.5-kb fragment and is deleted in M. bovis BCG.The constructed sets of LAMP primers are shown as lines and boxes. The segment of the RD1 sequence, depicted as solid-white boxes and named RD1, is inserted within M. tuberculosis, M. bovis, and M. africanum sequences. Asterisks show the specific conserved sequences within the M. tuberculosis complex.
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pone.0133759.g001: Alignment of the sequences including RD1, which encodes a 9.5-kb fragment and is deleted in M. bovis BCG.The constructed sets of LAMP primers are shown as lines and boxes. The segment of the RD1 sequence, depicted as solid-white boxes and named RD1, is inserted within M. tuberculosis, M. bovis, and M. africanum sequences. Asterisks show the specific conserved sequences within the M. tuberculosis complex.

Mentions: The oligonucleotide LAMP primers for specific detection of the M. bovis BCG genes were designed based on the RD1 sequence (GenBank accession no. AP010918.1). Genbank sequences initially considered were tested in silico through BLAST searches and alignment analysis [29]. Several candidate primer sets were suggested via online LAMP primer design software (PrimerExplorer 4, http://primerexplorer.jp/index.html; Eiken Chemical, Tokyo, Japan), and further refinement of primer design was developed manually based on the criteria described in “A Guide to LAMP primer designing” (http://primerexplorer.jp/e/v4_manual/index.html). Each selected primer sequence is given in Table 1 and their positions are shown in Fig 1.


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

Kouzaki Y, Maeda T, Sasaki H, Tamura S, Hamamoto T, Yuki A, Sato A, Miyahira Y, Kawana A - PLoS ONE (2015)

Alignment of the sequences including RD1, which encodes a 9.5-kb fragment and is deleted in M. bovis BCG.The constructed sets of LAMP primers are shown as lines and boxes. The segment of the RD1 sequence, depicted as solid-white boxes and named RD1, is inserted within M. tuberculosis, M. bovis, and M. africanum sequences. Asterisks show the specific conserved sequences within the M. tuberculosis complex.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514781&req=5

pone.0133759.g001: Alignment of the sequences including RD1, which encodes a 9.5-kb fragment and is deleted in M. bovis BCG.The constructed sets of LAMP primers are shown as lines and boxes. The segment of the RD1 sequence, depicted as solid-white boxes and named RD1, is inserted within M. tuberculosis, M. bovis, and M. africanum sequences. Asterisks show the specific conserved sequences within the M. tuberculosis complex.
Mentions: The oligonucleotide LAMP primers for specific detection of the M. bovis BCG genes were designed based on the RD1 sequence (GenBank accession no. AP010918.1). Genbank sequences initially considered were tested in silico through BLAST searches and alignment analysis [29]. Several candidate primer sets were suggested via online LAMP primer design software (PrimerExplorer 4, http://primerexplorer.jp/index.html; Eiken Chemical, Tokyo, Japan), and further refinement of primer design was developed manually based on the criteria described in “A Guide to LAMP primer designing” (http://primerexplorer.jp/e/v4_manual/index.html). Each selected primer sequence is given in Table 1 and their positions are shown in Fig 1.

Bottom Line: 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.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 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.

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 × 10(3) 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 × 10(3) 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.

No MeSH data available.


Related in: MedlinePlus