RESUMO
CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5' ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism - single crossing CPA - and provide details on alternative mechanisms.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Moldes GenéticosRESUMO
Chronic hepatitis B virus (CHBV) infection causes cirrhosis and hepatocellular carcinoma. Lamivudine (LAM) has been successfully used to treat CHBV infections but prolonged use leads to the emergence of drug-resistant variants. This is primarily linked to a mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV polymerase gene at position 204. Rapid diagnosis of drug-resistant HBV is necessary for a prompt treatment response. Common diagnostic methods such as sequencing and restriction fragment length polymorphism (RFLP) analysis lack sensitivity and require significant processing. The aim of this study was to demonstrate the usefulness of a novel diagnostic method that combines polymerase chain reaction (PCR), ligase detection reaction (LDR) and a nucleic acid detection strip (NADS) in detecting site-specific mutations related to HBV LAM resistance. We compared this method (PLNA) to direct sequencing and RFLP analysis in 50 clinical samples from HBV infected patients. There was 90% concordance between all three results. PLNA detected more samples containing mutant variants than both sequencing and RFLP analysis and was more sensitive in detecting mixed variant populations. Plasmid standards indicated that the sensitivity of PLNA is at or below 3,000 copies per ml and that it can detect a minor variant at 5% of the total viral population. This warrants its further development and suggests that the PLNA method could be a useful tool in detecting LAM resistance.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Lamivudina/uso terapêutico , Reação em Cadeia da Polimerase/métodos , Inibidores da Transcriptase Reversa/uso terapêutico , Antivirais/uso terapêutico , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos , Reação em Cadeia da Ligase , Mutação/efeitos dos fármacos , Polimorfismo de Fragmento de Restrição , Fitas Reagentes , Sensibilidade e EspecificidadeRESUMO
Cross-priming amplification (CPA) technology for tuberculosis diagnosis from sputum specimens was evaluated. The sensitivity of CPA from smear- and liquid culture-positive specimens was 96.9%, and that from smear-negative and liquid culture-positive specimens was 87.5%. The specificity of CPA in culture-negative specimens was 98.8%.
