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1. Introduction
Nucleic acid amplification tests are core technologies of clinical diagnosis. In pulmonary tuberculosis, such testing is capable of identifying Mycobacterium species in clinical respiratory samples more rapidly and accurately than sputum specimen examinations and culture-based methods. This advantage is key to appropriate treatment, prevention, and control of transmission of tuberculosis. In HIV detection, the nucleic acid amplification test is more sensitive and quantitative than other methods based on HIV-1-specific antibody or viral antigens, enabling the detection of HIV-1 at the initial stage of infection and the monitoring of disease progression (1,2).
Various nucleic acid amplification technologies have been devised, but the most widely used is PCR. In basic research, most researchers use PCR primarily for amplification, possibly because primer design is convenient and the enzymes are available at a reasonable price (3). In clinical diagnosis, on the other hand, isothermal nucleic acid amplification methods such as nucleic acid sequence-based amplification (NASBA) (4), strand displacement amplification (SDA) (5), rolling circle amplification (RCA) (6), helicase-dependent isothermal DNA amplification (HAD) (7), and loop-mediated isothermal amplification (LAMP) (8) are also used. The advantage of isothermal amplifications over PCR is that they do not require a complex device such as thermal cycler, improving throughput in situations when large numbers of clinical samples must be processed, as well as facilitating point-of-care diagnosis (4-9).
The performance of a nucleic acid amplification test depends largely on the performance of the enzymes involved. Thermostable DNA polymerase, first identified in Thermus aquaticus (Taq) in 1976 (10), has become widely used since the discovery of PCR. Concerning performances of Taq polymerase, it was initially reported that the activity decreased to 50% at incubation at 95°C for 1.6 h; the rate of processing was 60-150 nucleotides/sec; and the error rate was 0.38−1.32×104 errors/base (11). Since then, the performances of Taq polymerase were improved by genetic engineering. For example, the mutation of Phe667 into Tyr increased its efficiency of incorporation with ddNTP by 103-fold (12), and fusion of the helix-hairpin-helix motifs of DNA topoisom-erase V to Taq polymerase increased the enzyme's stability and processivity (13). The performances of DNA polymerases from the hyperthermophilic archaeon Thermococcus koda- karensis (KOD) or Pyrococcus furiosus(Pfu) and that from thermophilic bacteria Thermus thermophilus