The Hepatitis B virus (HBV) causes liver infection, but for some people, it is just an acute or short-term infection, however, for others, it can become a long-term, chronic infection called chronic hepatitis.
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Chronic Hepatitis B can lead to serious health issues. Vaccination against HBV is the best way to prevent infections with HBV. However, infections with HBV remain a top health problem worldwide. In 2013 alone, HBV infections caused approximately 600,000 deaths. According to Papastergiou et al., more than 350 million people are chronically infected with HBV.
Detection and measurements of HBV DNA levels are now routinely used for the identification of infectious, chronic carriers and the prediction and monitoring of efficacies of antiviral treatment.
Real-time PCR (RT PCR) is now widely accepted as the gold standard for quantification of viral nucleic acids. RT PCR allows quantification of HBV DNA with improved speed of detection, high sensitivity, and reproducibility, as well as a low risk of contamination. Several real-time PCR assays have been developed usually using two pairs of primers and probes often based on the conserved S and C regions of the HBV genome.
Primers used for the amplification of HBV complete genomes
|
Primera
|
Sequence (5′–3′)
|
Position (nt)b
|
Amplicon (bp)
|
Sequencing primer
|
|
PR1a
|
(Ta = 4°C)
|
|
|
|
|
251F
|
GACTYGTGGTGGACTTCTC
|
251–269
|
940
|
Yes
|
|
1190R
|
TCAGCAAAYACTYGGCA
|
1190–1174
|
|
Yes
|
|
PR1b
|
(Ta = 54°C)
|
|
|
|
|
595F
|
CACHTGTATTCCCATCCCA
|
595–613
|
1,203
|
Yes
|
|
1797R
|
CCAATTTMTGCYTACAGCCTC
|
1797–1777
|
|
No
|
|
1190F
|
AYGCAACCCCCACTGG
|
1190–1205
|
|
Yes
|
|
PR 2a
|
(Ta = 50°C)
|
|
|
|
|
2300F
|
CCACMWAATGCCCCTATC
|
2300–2317
|
1,131
|
Yes
|
|
215R
|
AGRAAMACMCCGCCTGT
|
215–200
|
|
Yes
|
|
PR 2b
|
(Ta = 50°C)
|
|
|
|
|
2819F
|
ACCWTATWCYTGGGAACAA
|
2819–2837
|
1,032
|
Yes
|
|
617Rc
|
GAYGAYGGGATGGGAATACA
|
617–598
|
|
Yes
|
|
654R
|
GSCCCAMBCCCATAGG
|
652–637
|
|
No
|
|
PR 3
|
(Ta = 52°C)
|
|
|
|
|
1859F
|
ACTNTTCAAGCCTCCRAGCTG
|
1859–1879
|
959
|
No
|
|
1877F
|
CTGTGCCTTGGRTGGCTT
|
1894–1877
|
|
Yes
|
|
2835R
|
GTTCCCAVGWATAWGGTGAYCC
|
2835–2814
|
|
Yes
|
|
PR 4
|
(Ta = 57°C)
|
|
|
|
|
1584F
|
ACTTCGMBTCACCTCTGCACGT
|
1583–1604
|
748
|
No
|
|
2331R
|
GGAAGYGTKGAYARGATAGGGGCATT
|
2331–2306
|
|
Yes
|
|
2396R
|
GTCKGCGAGGYGAGGGAGTT
|
2396–2377
|
|
No
|
aSuffix “F” indicates forward primer, whereas suffix “R” indicates reverse primer.
bWith reference to NC_003977. https://www.ncbi.nlm.nih.gov/nuccore/NC_003977 Hepatitis B virus (strain ayw) genome
cAn annealing temperature of 54°C was used during sequencing cycling to ensure a high rate of success of sequencing.
BNA Primers and Probes
Artificial nucleic acids such as bridged nucleic acids (BNAs) can also be incorporated into oligonucleotide probes to increase sensitivity and selectivity of the probes.
|
Primer
Probes
|
Sequence
|
Bases
|
Tm (°C)
|
|
|
|
|
|
|
P1
|
GACCACCAAATGCCCCTAT
|
19
|
55
|
|
P2
|
CCRAGAYYGAGATCTTCTGCGAC
|
23
|
53
|
|
BNA probe
|
FAM-TCGTCTAACAACAGT-BHQ1
|
|
|
|
TaqMan
|
FAM-TCGTCTAACAACAGT(TAMMRA)AGTTTCCGGAAGTGT-P
|
|
|
aThe underlined nucleotides indicate a BNA monomer substitution. Tm, temperature; BNA, bridged nucleic acid (e.g. BNA or LNA) ; HBV, hepatitis B virus; PCR, polymerase chain reaction. (Source: Wang et al. 2011.) https://www.cdc.gov/hepatitis/hbv/index.htm
More primers and probe sequences for the S and C regions.
|
Primer and Probe
|
Sequence (5′-3′)
|
|
S-F
|
GATGTGTCTGCGGCGTTTTA
|
|
S-R
|
GCAACATACCTTGATAGTCCAGAAGAA
|
|
S-P
|
Vic-CCTCTICATCCTGCTGCTATGCCTCA-BHQ1
|
|
C-F
|
TTCCGGAAACTACTGTTGTTAGAC
|
|
C-R
|
ATTGAGATTCCCGAGATTGAGA
|
|
C-P
|
Fam-CCCTAGAAGAAGAACTCCCTCGCCTC-BHQ1
|
|
Sa-F
|
TCGTGTTACAGGCGGGGTTT
|
|
Sa-R
|
GGCACTAGTAAACTGAGCCA
|
|
Ca-F
|
CCTACTGTTCAAGCCTCCAA
|
|
Ca-R
|
AATGTCCTCCTGTAAATGAATGT
|
Reference
Chao Liu, Le Chang, Tingting Jia, Fei Guo, Lu Zhang, Huimin Ji, Junpeng Zhao and Lunan Wang; Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.Virology Journal201714:94. https://doi.org/10.1186/s12985-017-0759-8.
Chook, J. B., Teo, W. L., Ngeow, Y. F., Tee, K. K., Ng, K. P., & Mohamed, R. (2015). Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G. Journal of clinical microbiology, 53(6), 1831-5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432068/
Hepatitis B Info:
https://health.mil/Military-Health-Topics/Health-Readiness/Immunization-Healthcare/Vaccine-Preventable-Diseases/Hepatitis-B
http://www.medsci.org/v08p0321.htm
https://virologyj.biomedcentral.com/articles/10.1186/s12985-017-0759-8
Papastergiou, V., Lombardi, R., MacDonald, D. et al. Global Epidemiology of Hepatitis B Virus (HBV) Infection. Curr Hepatology Rep (2015) 14: 171. https://doi.org/10.1007/s11901-015-0269-3. https://link.springer.com/article/10.1007%2Fs11901-015-0269-3
Wang, Q., Wang, X., Zhang, J., & Song, G. (2011). LNA real-time PCR probe quantification of hepatitis B virus DNA. Experimental and therapeutic medicine, 3(3), 503-508. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438541/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3729363/
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