Tetsu Hase

Operational Feasibility of Using Loop-Mediated Isothermal Amplification for Diagnosis of Pulmonary Tuberculosis in Microscopy Centers of Developing Countries

ABSTRACT The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified manual DNA extraction was evaluated for accuracy and ease of use. The sensitivity of LAMP in smear- and culture-positive sputum specimens was 97.7% (173/177 specimens; 95% confidence interval [CI], 95.5 to 99.9%), and the sensitivity in smear-negative, culture-positive specimens was 48.8% (21/43 specimens; CI, 33.9 to 63.7%). The specificity in culture-negative samples was 99% (500/505 specimens; CI, 98.1 to 99.9%). The average hands-on time for testing six samples and two controls was 54 min, similar to that of sputum smear microscopy. The optimal amplification time was 40 min. No indeterminate results were reported, and the interreader variability was 0.4%. Despite the use of a single room without biosafety cabinets for all procedures, no DNA contamination was observed. The assay was robust, with high end-point stability and low rates of test failure. Technicians with no prior molecular experience easily performed the assay after 1 week of training, and opportunities for further simplification of the assay were identified.

Transcription of a region downstream from λ ori is required for replication of plasmids derived from coliphage lambda

SummaryDNA replication of lambda phage depends on transcriptional activation at or around the λori region by RNA polymerase. To elucidate the function of the transcriptional activation, we constructed several plasmids carrying λori and lacP, whose relative locations and directions were different from each other, and studied replication activity of these recombinant plasmids. Transcription in a region immediately downstream from λori, but not in the λori region, was found to be essential for plasmid replication. Transcription proceeding over a certain minimal length was required and only rightward-directed transcription was effective for the activation

Analysis of promoters of adenovirus type 12 E1A gene in a cell-free transcription system

Transcription from the E1A gene of adenovirus type 12 initiates at two sites in vivo. In this paper we analyzed the E1A promoter(s) in a cell-free transcription system. Primer extension assay revealed that transcriptions were accurately initiated at two sites apart by 140 bp. The efficiency of transcriptions from two sites was almost equal in the standard reaction. However, when transcription reaction was done following pre-incubation of a nuclear extract and template DNA in the absence of ribonucleotides, transcription from the site proximal to E1A gene significantly decreased with little effect on that from distal site. This suggests the existence of a mechanism which controls the efficiency of transcriptions from two start sites of E1A gene.

Biological characteristics of plasmid carrying a repeated deoxyribonucleic acid sequence

It was found that a plasmid which had a foreign deoxyribonucleic acid (DNA) between two repeated sequences did not multiply in E. coli recBCsbcB, even if it multiplied in wild-type E. coli, E. coli recBC or E. coli recBCsbcBrecF when the insert was longer than 351 base pair. The multiplication of these plasmids were, however, inhibited when a plasmid expressing recF gene was introduced into E. coli recBCsbcBrecF. The inviability of the plasmid carrying the repeated sequence in E. coli recBCsbcB was discussed by the mechanism of recombination, and the functions of recF, recBC and sbcB were speculated. When E. coli recBC was transformed with pDR1 which was a derivative of pBR322 carrying a directly repeated sequence between which a DNA fragment derived from plasmid R6K with its origin was inserted, the intramolecular recombinant appeared. The recombinant recovered was, however, only the plasmid which had the replication origin of pBR322. The result suggests that pBR322 is compatible with pDR1 but R6K is not. The replication origin of R6K seems to be preferrentially used by pDR1.

Mutants of Shigella sonnei Deficient in DNA Polymerase I

Mutants of Shigella sonnei (S. sonnei) deficient in DNA polymerase I were isolated after mutagenesis with nitrosoguanidine. The isolation of the mutants was facilitated by the use of a strain harboring plasmid pBR313 which required DNA polymerase I for its muliplication. The mutants isolated could not maintain the plasmid and became sensitive to methyl methanesulfonate (MMS) and to ultraviolet light (UV) irradiation. Assays performed on crude extracts established that the mutants were deficient in an enzyme with DNA polymerase activity. All of these properties are the same as those of E. coli polA. Several MMS-resistant revertants isolated from one of the S. sonnei polA mutants regained 3-120% of the DNA polymerase activity found in the extracts of the wild-type parent strain. Most though not all of the revertants could support the multiplication of plasmid pBR313.