title: 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 × 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.
authors.affiliation: Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kouzaki
authors.affiliation: Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Sasaki
authors.affiliation: Department of Pediatrics, National Defense Medical College, Saitama, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Tamura
authors.affiliation: Department of Laboratory Medicine, National Defense Medical College Hospital, Saitama, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Hamamoto
authors.affiliation: Department of Laboratory Medicine, National Defense Medical College Hospital, Saitama, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yuki
authors.affiliation: Department of Urology, National Defense Medical College, Saitama, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Sato
authors.affiliation: Department of Global Infectious Diseases and Tropical Medicine, National Defense Medical College, Saitama, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Miyahira
authors.affiliation: Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, Saitama, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kawana
mesh_terms: D001500:BCG Vaccine / Q000009:adverse effects; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D001826:Body Fluids / Q000382:microbiology; D003124:Colorimetry; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D003937:Diagnosis, Differential; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics* / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation; D000067716:Point-of-Care Testing; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013696:Temperature; D014376:Tuberculosis / Q000175:diagnosis / Q000209:etiology / Q000382:microbiology*
chemical_list: D001500:BCG Vaccine; D017931:DNA Primers; D004269:DNA, Bacterial