Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Zeldis, Jerome B.; Lee, Jae Hag; Mamish, D.; Finegold, D; Sircar, R.; Ling, Qihe; Knudsen, Peter Juel Thiis; Kuramoto, I. K.; Mimms, Larry

doi:10.1172/jci114326

Cited by 46 publications

(14 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such fluids include urine, (Lz) serum, (3)(4)(5) feces, (6'7) amniotic fluid, (8) and cerebrospinal fluid. (8) When detection or quantification of microbial genomes from clinical specimens is desired, the presence of PCR inhibitory factors potentially leads to false-negative results or underestimations, respectively.…”

mentioning

confidence: 99%

Inhibitory effect of salivary fluids on PCR: potency and removal.

Ochert

Boulter²,

Birnbaum³

et al. 1994

Genome Research

View full text Add to dashboard Cite

Various body fluids contain agents that inhibit the PCR. Such fluids include urine, (Lz) serum, (3-5) feces, (6'7) amniotic fluid, (8) and cerebrospinal fluid. (8) When detection or quantification of microbial genomes from clinical specimens is desired, the presence of PCR inhibitory factors potentially leads to false-negative results or underestimations, respectively.We have been engaged in PCR detection and quantification of the EpsteinBarr virus (EBV) genome in oral fluids. Preliminary attempts to demonstrate viral DNA in whole mouth fluid (WMF) and parotid saliva (PS) from study subjects resulted in poor positivity yields. This was despite having established PCR conditions for the sensitive detection of EBV derived from cell culture and of plasmids that contain subgenomic EBV inserts. These results were surprising in view of previous reports that the virus could be isolated from saliva at high rates by the lymphocyte transformation assay. (9'1~ We therefore investigated the possibility that saliva might also contain PCR inhibitory factors. We demonstrate here that saliva potently inhibits PCR and describe a simple procedure to eliminate this effect. MATERIALS AND METHODSWMF was collected from 10 healthy human subjects after mechanical stimulation by the chewing of sterile rubber bands. PS was obtained by applying a Lashley cup to Stensens' duct; stimulation was achieved by placing citric acid crystals on the tongue at -30-sec intervals. WMF collection preceded PS collection. WMF was clarified, passed through 5.0-1~m Acrodisc filters, aliquoted, and stored at -70~ until use. PS samples were passed through 0.45-1~m filters, similiarly aliquoted, and frozen.Plasmid pBR322 containing the nonreiterated BamHI K fragment of the B95-8 strain of EBV (11) was used as the principal source of DNA template for PCR. Oligonucleotide PCR primers and probe used, respectively, for the amplification and detection of the BamH! K region of EBV were synthesized in an Applied Biosystems 380B synthesizer; the sequences of these oligomers have been reported previously. (~2) PCR was performed in a Perkin-Elmer Cetus PCR DNA thermal cycler. Thirty-five cycles were performed per run. Cycling conditions were as follows: denaturation at 94~ for 1 min; annealing at 50~ for 40 sec; and extension at 72~ for 1 min.WMF and PS samples, individually spiked with plasmid DNA, underwent each of the following DNA extraction procedures: Geneclean II (Stratech Scientific, Luton, UK); Magic DNA Clean-up System (Promega, Madison, WI); Sephadex G-50 chromatography; phenol-chloroform; guanidinium thiocycanate/silica with or without proteinase K pretreatment; microwaving; boiling for 5 min; and Chelex-100. The Geneclean and Magiprep methods were performed according to manufacturers' instructions. G-50 chromatography was performed as described by de Franchis et al. (13) Extraction with phenol-chloroform was performed as follows: Each 100-1~1 WMF or PS sample was vortexed with an equal volume of 25:24:1 phenol/chloroform/ isoamyl alcohol and spun briefly; t...

show abstract

mentioning

confidence: 99%

Inhibitory effect of salivary fluids on PCR: potency and removal.

Ochert

Boulter²,

Birnbaum³

et al. 1994

Genome Research

View full text Add to dashboard Cite

show abstract

“…'5216 HBV DNA is detected in HBsAg, anti-HBe positive chronic carriers17 as well as in some individuals long after recovery from acute hepatitis B.18 Highly sensitive but until now merely qualitative detection of HBV DNA has become possible by use of polymerase chain reaction (PCR). 13,[19][20][21][22] Due to lack of standardization, these tests have a highly varying sensitivity and are accompanied by a risk of false r e s~l t s . '~,~~…”

mentioning

confidence: 99%

Quantification of hepatitis B virus DNA over a wide range from serum for studying viral replicative activity in response to treatment and in recurrent infection

Ranki

Schätzl²,

Zachoval³

et al. 1995

Hepatology

View full text Add to dashboard Cite

A new standardized test for hepatitis B virus (HBV) DNA with increased sensitivity and range over previous assays (30 to 10(6) HBV genomes/test) was evaluated in this study. The quantitative results from the test have been validated using international reference specimens of known titer and a reference solution hybridization test. The test has small variability considering the wide dynamic range. The CV was 14% within one experiment and 32% to 39% between independent experiments. Hepatitis B surface antigen (HBsAg)-negative, anti-HBc-positive blood donor sera (n = 25) were all negative for HBV DNA in the new test, whereas 63% (n = 19) of HBsAg-positive healthy carriers had measurable quantities of HBV DNA. In five example cases of chronic hepatitis B patients responding to alfa-interferon treatment but remaining virus positive, HBV DNA was consistently present in posttreatment sera in a titer range 4 x 10(3) to 10(6)/mL not detectable by the conventional hybridization test. In two complete responders, the HBV DNA titer decreased over six orders of magnitude to below cutoff of the test. In four liver transplant recipients with chronic hepatitis B, viral recurrence was detected by the new test at an early stage much before the clinical relapse. Unlike serology, the test was suitable also in patients under anti-HBs immunoprophylaxis. In conclusion, the new colorimetric polymerase chain reaction (PCR) test allowed thousandfold increased sensitivity in quantification of HBV DNA in patient sera. The test may have future applications in improving assessment of efficacy of antiviral treatment and guiding therapeutic interventions.

show abstract

“…Therefore, PCR is the preferred test for detection of HBV DNA sequences in the clinical specimens for diagnostic purposes [14]. However, the efficacy of PCR amplification may be affected by the presence of number of Taq polymerase inhibitors in the serum [7,23] and thus DNA extraction is required before amplification.…”

Section: Discussionmentioning

confidence: 99%

An improved method for the isolation of hepatitis B virus DNA from human serum

Changotra

Sehajpal

2013

Indian J. Virol.

View full text Add to dashboard Cite

Studies show that hepatitis B virus (HBV) DNA isolation methods vary in their efficiency to extract DNA from serum samples. The purpose of the present study was to develop an improved method for isolation of HBV DNA and compare it with commonly used HBV DNA isolation protocols. In order to develop HBV DNA isolation protocol, serum samples were collected from patients and screened for the presence of hepatitis B surface antigen, hepatitis B e antigen and HBV DNA. Highly viremic samples were pooled and used to compare commonly used HBV DNA isolation methods; namely alkaline lysis, microwave treatment, organic, inorganic with modified inorganic method. DNA isolated by these methods was detected qualitatively by polymerase chain reaction and quantitatively with competitive polymerase chain reaction (cPCR). The modified inorganic method gave maximum yield of HBV DNA followed by inorganic, organic, microwave treatment and alkaline lysis method. Our data also demonstrated a critical role of proteinase K in HBV DNA isolation. DNA isolation method described here, in combination with a reproducible and sensitive quantitative technique would further help in accurate classification of HBV infected patients, designing suitable drug regimen for treatment and monitoring antiviral treatment as well as emergence of drug resistant mutants.

show abstract

Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Cited by 46 publications

References 17 publications

Inhibitory effect of salivary fluids on PCR: potency and removal.

Inhibitory effect of salivary fluids on PCR: potency and removal.

Quantification of hepatitis B virus DNA over a wide range from serum for studying viral replicative activity in response to treatment and in recurrent infection

An improved method for the isolation of hepatitis B virus DNA from human serum

Contact Info

Product

Resources

About