RESUMEN
Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.
Asunto(s)
Hepacivirus/genética , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Bases , Donantes de Sangre , Cartilla de ADN/genética , ADN Viral/genética , Genotipo , Anticuerpos contra la Hepatitis B/sangre , Humanos , Tamizaje Masivo , Control de Calidad , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y EspecificidadRESUMEN
The complexity of Nucleic acid Amplification Technology (NAT(1)), comprising sample preparation, amplification and detection methods, requires specific design considerations for both the laboratory and the procedures utilized in such testing. The purpose of this paper is to establish technical considerations for the performance of NAT. These include the collection, handling and assay of specimens and the design of laboratories to routinely and reliably detect low levels of nucleic acid sequences. The sensitivity of NAT due to the exponential amplification of nucleic acids makes contamination a major concern from specimen collection to sample detection. Therefore, laboratories need to be designed to prevent and control contamination through adequate equipment and appropriate workflow. These technical considerations should provide a basis for establishing a robust and reproducible NAT system.