Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63°C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.Because of their slow growth rate, identification of mycobacteria is a notorious problem for public health and clinical laboratories. To address the need for rapid and sensitive identification of Mycobacterium tuberculosis and other mycobacteria, various genotyping methods for routine diagnosis have been introduced during the past decade (1,2,4,9,10,(21)(22)(23)25). The recent trend in genetic testing is to make systems fully automatic with high-throughput analysis. Although this may be an ideal approach, it requires expensive equipment as well as a huge amount of space in routine diagnostic laboratories. As a result, it centralizes genetic testing in highly sophisticated facilities. If this trend continues, escalated pressure for transportation of clinical specimens will pose another problem. On the other hand, there are pressing needs for point-of-care testing at hospitals and primary care facilities.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method which relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment (13,(15)(16)(17)...

ABSTRACT. It has been often found that the provisionized Japanese monkeys here limb-malformed babies since 1955 in Japan. We reported that some organochlorin pesticide residues might have related to the cause of the malformation of limbs. It was found that the tissues of the dead Japanese monkeys were contaminated with Heptachlor epoxide, Dieldrin, BHC, DDT, and Chlordane isomers. The concentrations of Heptachlor epoxide in the malformed monkeys were significantly higher (kidney, p<0.05) than those in the normal monkeys. The levels of Heptachlor epoxide (kidney) and Dieldrin (liver) in the adult female monkeys who had borne malformed babies were also significantly higher (p<0.05) than those in the adult females who had not.Relation of the Congenital Limb-malformation in the Japanese Monkeys and Organochlorin Pesticides

Peptide nucleic acid-mediated competitive PCR clamping, which can selectively amplify mutant alleles, was developed to detect mutations in four codons (513, 516, 526, and 531) of the rpoB gene in Mycobacterium tuberculosis strains. This simple method successfully identified the mutations in all 40 of the M. tuberculosis strains tested.

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Recent increases in infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria are a serious hazard to public health (2,6,(11)(12)(13)20). More than 30 mycobacterial species are recognized as pathogenic or potentially pathogenic. Mixed infections with two or more species (1,18,29,31) and nosocomial infections (3,8,19,25,27) are always troublesome. Therefore, timely identification of causative mycobacteria is imperative for appropriate therapy and epidemiological analyses.Genotyping the sequence polymorphisms in the genus Mycobacterium can identify species quickly and unequivocally. PCR-restriction fragment polymorphism analysis (PRA) of the hsp65 gene (4, 21, 22, 28), 16S ribosomal DNA (rDNA) (5, 14, 15, 30), 16S-23S rDNA internal transcribed spacer (ITS) (23), dnaJ (26), rpoB (17), and gyrB (16) has been intensively studied. However, these methods are not applicable for a polymycobacterial sample because too many restriction fragments from multiple species of mycobacteria in it blur the analysis.We developed a fluorescence capillary electrophoresis (FCE)-based restriction fragment-single-strand conformation polymorphism (FCE-RF-SSCP) technique which can simultaneously identify multiple mycobacterial species in a sample. We first established an algorithm for species identification by applying this technique to 110 defined mycobacterial strains.The technique was further validated on "spiked" specimens, clinical sputum specimens, and sputum obtained from a patient with mixed M. avium and M. intracellulare infection.