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 kvantificering af hepatitis 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: Fremskridt og udfordringer inden for detektion af hepatitis B-virus 

Polymerasekædereaktionsbaseret påvisning og kvantificering i realtid af hepatitis 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  

Kvantificering af hepatitis B-virus (HBV) DNA i humane serumprøver ved hjælp af PCR i realtid

Til at begynde med skal du optø alle reagenser, der leveres i PCR-sættet i realtid ved stuetemperatur. Når de er optøet, pulser hvirvelstrømmen i 10 sekunder med moderat hastighed for at blande dem. Derefter centrifugeres ved 8.000 G i 10 sekunder ved stuetemperatur.Opbevar de optøede komponenter på en køleblok. Forbered derefter en PCR-blanding til en HBV-positiv prøve, fem HBV-standarder og ingen skabelonkontrol. Bland grundigt de tilsatte reagenser ved forsigtigt pipettering syv til 10 gange, efterfulgt af pulshvirvelstrøm, og centrifuger ved 8.000 G i 10 sekunder ved stuetemperatur.Derefter pipetteres 15 mikroliter PCR-blanding i hvert PCR-rør. Tilsæt 15 mikroliter ekstraheret DNA-prøve, fem HBV-standarder og nukleasefrit vand som en ingen skabelonkontrol. Luk PCR-rørene, og drej centrifugen kortvarigt.Læg PCR-rørene i en PCR-maskine i realtid. Start nu den termiske cyklersoftware. Vælg fluorescerende farvestoffer, og programmer den termiske cyklist til PCR i realtid.Efter kørsel skal du analysere forstærkningsplottet ved hjælp af en lineær skala. Indstil PCR-reaktionstærsklen inden for eksponentialfasen. Oprethold konsistens i tærskelindstillingerne for nøjagtighed og reproducerbarhed.Brug den samme tærskel for alle prøver på en bestemt PCR-maskine. Overvej prøver med amplifikation før CD-værdi 38 som HBV-positive baseret på analysens cutoff. Brug fem kvantificeringsstandarder for HBV-specifikt DNA, kalibreret mod WHO's fjerde internationale standard for HBV-DNA.Generer en standardkurve med disse standarder for at kvantificere HBV-DNA i ukendte prøver. Fortolk kun værdier for ukendte prøver, hvis standardkurvehældningen er mellem minus 3,1 og minus 3,6, R-kvadratværdien er mellem 0,99 og 1,0, PCR-effektiviteten er inden for 90% til 110%og der er ingen forstærkning i den negative kontrol.