Name: Quantifying Hepatitis B Virus (HBV) DNA in Human Serum Samples Using Real-Time PCR
Uploaded: 2023-12-15T14:50:00
Description: Watch this Scientific Journal Video about Quantifying Hepatitis B Virus HBV DNA in Human Serum Samples Using Real-Time PCR at JoVE.com

quantifying hepatitis b virus (hbv) dna in human serum samples using real-time pcr

Påvisning og kvantifisering av hepatitt B-virus-DNA i humant serum

Hepatitis B virus (HBV) is a partially double-stranded DNA virus from the genus Orthohepadnavirus and the Hepadnaviridae family1. It can trigger a chronic infection that persists throughout one&#39;s life, potentially leading to liver cirrhosis and hepatocellular carcinoma2,3,4. According to the World Health Organization (WHO), an estimated population of 296 million people were living with chronic hepatitis B infection in 2019, and 1.5 million people were newly infected each year5.
The identification and measurement of HBV DNA in serum or plasma samples serve as a valuable method for detecting individuals with an ongoing hepatitis B infection, assessing the efficacy of antiviral treatment, and predicting the probability of treatment success6,7,8,9,10,11. High viral load is associated with an increased risk of liver disease progression, including cirrhosis and liver cancer12,13. Hence, precise measurement of viral load is crucial for tracking the progression of HBV infection and informing decisions regarding treatment.
Quantitative real-time PCR assays have higher sensitivity, broader dynamic range, and more accurate quantification of HBV DNA than conventional PCR techniques14,15,16,17. Several commercial real-time PCR-based molecular diagnostic kits for the quantitation of HBV DNA in serum or plasma samples are available in the market. Here, we describe a detailed workflow for the detection and quantification of HBV DNA in human serum or plasma using a commercially available, IVD-marked real-time PCR-based kit. The kit is a widely used18,19, low-cost, yet highly sensitive assay, and its performance characteristics are comparable with other commercially available CE-marked kit(s)18. Apart from the HBV target region amplification, an endogenous internal control gene is also included in the kit to verify the quality of samples, quality of extracted DNA, PCR amplification, and possible PCR inhibition.

Forfatter Spotlight: Fremskritt og utfordringer innen hepatitt B-virusdeteksjon 

Sanntids polymerasekjedereaksjonsbasert deteksjon og kvantifisering av hepatitt B-virus DNA

In this video, we will show you a step-by-step protocol to detect HBV DNA and quantify its viral load using our IVD approved real-time PCR waste, through PCR HBV viral load kit. HBV diagnosis research uses, real time PCR, digital PCR, NGS and serological tests. Realtime PCR is the gold standard for HBV DNA detection and viral load quantification.Digital PCR quantifies absolute viral load. NGS identifies new mutations and serological tests detect antigens or antibodies. There are several experimental challenges with detecting low viral load cases and occult HBV infections.Some assays may not fall from well across all HBV genotypes and emerging new mutations may lead to false negative results. Compared to other techniques the realtime PCR based protocol is the most sensitive, specific and quantitative method available for HBV detection. It is also a relatively fast technique, which makes it ideal for clinical use.

Watch this Scientific Journal Video about Quantifying Hepatitis B Virus HBV DNA in Human Serum Samples Using Real-Time PCR at JoVE.com

Quantifying Hepatitis B Virus (HBV) DNA in Human Serum Samples Using Real-Time PCR  

Kvantifisering av hepatitt B-virus (HBV) DNA i humane serumprøver ved bruk av sanntids PCR

For å begynne, tine alle reagenser som følger med i sanntids PCR-settet ved romtemperatur. Når de er tint, pulsvirvler komponentene i 10 sekunder med moderat hastighet for å blande dem. Deretter sentrifuge ved 8.000G i 10 sekunder ved romtemperatur.Oppbevar de tinte komponentene på en kjøleblokk. Deretter forbereder du en PCR-blanding for en HBV-positiv prøve, fem HBV-standarder og ingen malkontroll. Bland de tilsatte reagensene grundig ved forsiktig pipettering syv til 10 ganger, etterfulgt av pulsvirvel og sentrifuge ved 8.000 G i 10 sekunder ved romtemperatur.Deretter blandes pipett 15 mikroliter av PCR-en inn i hvert PCR-rør. Legg til 15 mikroliter ekstrahert DNA-prøve, fem HBV-standarder og nukleasefritt vann som ingen malkontroll. Lukk PCR-rørene og spinn kort i sentrifugen.Legg PCR-rørene i en PCR-maskin i sanntid. Start nå programvaren for termisk sykling. Velg fluorescerende fargestoffer og programmer den termiske syklisten for sanntids PCR.Etter kjøring, analyser forsterkningsplottet ved hjelp av en lineær skala. Sett PCR-reaksjonsterskelen i eksponentiell fase. Oppretthold konsistens i terskelinnstillinger for nøyaktighet og reproduserbarhet.Bruk samme terskel for alle prøver på en bestemt PCR-maskin. Vurder prøver med amplifikasjon før CD-verdi 38 som HBV-positive basert på analysens cutoff. Bruk fem kvantifiseringsstandarder for HBV-spesifikt DNA, kalibrert mot den fjerde WHO-internasjonale standarden for HBV DNA.Generer en standardkurve med disse standardene for å kvantifisere HBV DNA i ukjente prøver. Tolk verdier for ukjente prøver bare hvis standard kurvehelling er mellom minus 3,1 og minus 3,6, R-kvadratverdien er mellom 0,99 og 1,0, PCR-effektiviteten er innenfor 90% til 110% og det er ingen forsterkning i den negative kontrollen.