Journal of Clinical Laboratory Analysis 4:479-482 (1990)
Detection of Hepatitis B Virus DNA Using the Polymerase Chain Reaction Technique Shuichi Kaneko,’ Kenichi Kobayashi,’ and Roger H. Miller2 ‘First Department of Internal Medicine, Kanazawa University, Kanazawa, Japan; 2Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland The polymerasechain reaction (PCR)technique has been utilized for the detection of hepatitis B virus (HBV) DNA, and several factors related to the selection of primer pairs for the PCR amplification have been demonstrated. The sensitivity of the PCR assay was compared with that of slot-blot hybridization for detecting HBV-DNA. Analysis by the PCR technique with Southern blot hybridization provided a > 104-foldincrease in sensitivity over the slot-blot hybridizationanalysis. Also, a rapid and sensitive PCR method for the detection of serum HBV-DNA was developed: HBV-DNA is released from virions by incubating serum with NaOH followed by neutralKey words:
ization with HCI. HBV-DNA sequences are then detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with successive sets of primer pairs. In testing serial samples from chimpanzees experimentally infected with HBV, HBV-DNA was detected 2-3 wk before the appearance of hepatitis surface antigen (HBsAg) and continued to be detectable for a short period after the production of antibody to HBsAg. Results from testing of human serum demonstrated that the majority of patients with HBsAg in serum had HBV-DNA as well and that some patients had HBV-DNA in serum in the absence of HBsAg.
HBsAg, HBeAg, DNA polymerase, acute hepatitis B, chronic hepatitis B, hybridization
INTRODUCTION The polymerase chain reaction (PCR) technique is a method for amplifying nucleic acid by repeated cycles of hightemperature denaturation, oligonucleotide primer hybridization, and polymerase extension. Previous investigations with this technique have shown amplification of > 105-fold over the input quantity of nucleic acid ( 1 ) . In the past, two methods were available for detecting the presence of hepatitis B virus (HBV) DNA in serum: the endogenous DNA polymerase reaction (2) and dot- or slot-blot hybridization analysis (3-8). Of the two, slot-blot hybridization was demonstrated to be more sensitive; it can detect HBV- DNA in amounts as low as approximately 0.1 pg, corresponding to approximately 3 x lo4 virus genomes (3,5,7). PCR amplification has been used for detecting serum HBVDNA, and the sensitivity of the PCR assay was compared with that of slot-blot hybridization (9-1 1). Also, a rapid and sensitive PCR method for the detection of serum HBV-DNA was developed ( 12).
length ( 1 3). Several factors related to the selection of primer pairs for HBV-DNA (Fig. 1 ) detection were summarized (14): (a) primers specific for the single-stranded (primer pair 6 and 7) or double-stranded (primer pair 1 and 3) regions of the HBV genome are equally effective in amplifying serum HBVDNA; although it is possible that the single-stranded regions of the HBV genome are not amplified optimally during the first round of PCR, there is no significant effect found at the end of 30 cycles of amplification; (b) maximum application is seen using primers separated by no more than 500 nucleotides (e.g., primer pairs 1 and 2, 1 and 3, 6 and 7); and (c) primers with up to 14%mismatch (3 on the 21 nucleotides or primer 21-3) are as effective as the perfectly paired primer (primer 21) at amplifying HBV-DNA in a PCR assay using annealing temperatures of 36°C or 60°C. Since HBV genome sequences rarely vary by more than 10% worldwide, it is likely that primer pairs complementary to most regions of the virus genome should be appropriate for use in PCR assays.
SELECTION OF PRIMERS Systematic evaluation of the characteristics of optimal primer pairs is necessary for efficient PCR amplification of HBV-DNA, which is a circular molecule approximately 3,200 nucleotides long with a single-stranded region of variable 0 1990 Wiley-Liss, Inc.
Received May 14, 1990; accepted May 26, 1990 Address reprint requests to Shuichi Kaneko, M.D., First Department of Internal Medicine, Kanazawa University, Takara-Machi 13- 1, Kanazawa 920, Japan.
480
Kaneko et al. RAPID AND SENSITIVE PCR METHOD
5
Contamination with small amounts of DNA represents a considerable problem because of the sensitivity of PCR analysis. To eliminate sources of contamination, reagents were aliquotted and reaction mixtures were prepared with the use of disposable syringes and pipets. All reagents were assayed for the presence of HBV-DNA. However, these precautions are sometimes not enough to eliminate contamination; therefore, a procedure that minimizes false-positive results is - v necessary. Also, although PCR-SBH offers a tremendous increase in the ability to detect serum HBV-DNA, it is timeconsuming, involves several critical steps, and utilizes a radiolabeled probe that must be prepared frequently. A senc sitive and rapid PCR method that reduces the possibility of contaminating samples with exogenous HBV-DNA was developed ( 12 ) . In the NaOH extraction method, a 10-kl aliquot of serum was pipetted into a 0.5-ml microcentrifuge tube and incubated in 0.1 M NaOH at 37°C for 60 min. The solution was neutralized with HCl and amplified by PCR. The NaOH extraction method was either as effective as or slightly more effective than the phenol chloroform extraction method for Fig. 1. Map of the HBV genome and primers. The circular, partially release of HBV-DNA from serum particles for amplification double-stranded genome of HBV is shown with primers 1-8,21,21-3, and in the PCR assay (Fig. 2 ) . The HBV-DNA produced in a PCR 21-6. The position and direction of primers are shown on the map by arrows. Primer pairs separated by 160 base pair (bp) (primers 1 and 2), 270 bp (primers reaction mixture could be reamplified if the second primer I and 3), 553 bp (primers I and 4), 1,135 bp (primers 1 and 5), 2,037 bp pairs, internal to the original pair, were used. When an am(primers 1 and 7), and 2,623 bp (primers 1 and 8) were used to amplify equiv- plified reaction mixture was reamplified with internal primalent amounts of recombinant HBV-DNA. Primer pairs specific to single- ers, a DNA band of expected size was detected in samples stranded (primers 6 and 7) and double-stranded(primers 1 and 3) regions were containing more than lo-' pg of HBV-DNA before the first used to amplify serumHBV-DNA. Primers consisting of 21 nucleotides with no template mismatch (primer 21) or with three (primer 21-3) or six (21-6) PCR amplification reaction (PCR-PCR technique). Thus, nucleotides mismatched were used in a PCR reaction with primer 3 to detect equivalent amounts of recombinant HBV-DNA of known nucleotide sequence.
1 2 3 4 5 6 7 8 9
ESTABLISHINGTHE SENSITIVITY OF THE PCR TECHNIQUE Slot- or dot-blot hybridization analysis can detect
Detection of Hepatitis B Virus DNA Using the Polymerase Chain Reaction Technique Shuichi Kaneko,’ Kenichi Kobayashi,’ and Roger H. Miller2 ‘First Department of Internal Medicine, Kanazawa University, Kanazawa, Japan; 2Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland The polymerasechain reaction (PCR)technique has been utilized for the detection of hepatitis B virus (HBV) DNA, and several factors related to the selection of primer pairs for the PCR amplification have been demonstrated. The sensitivity of the PCR assay was compared with that of slot-blot hybridization for detecting HBV-DNA. Analysis by the PCR technique with Southern blot hybridization provided a > 104-foldincrease in sensitivity over the slot-blot hybridizationanalysis. Also, a rapid and sensitive PCR method for the detection of serum HBV-DNA was developed: HBV-DNA is released from virions by incubating serum with NaOH followed by neutralKey words:
ization with HCI. HBV-DNA sequences are then detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with successive sets of primer pairs. In testing serial samples from chimpanzees experimentally infected with HBV, HBV-DNA was detected 2-3 wk before the appearance of hepatitis surface antigen (HBsAg) and continued to be detectable for a short period after the production of antibody to HBsAg. Results from testing of human serum demonstrated that the majority of patients with HBsAg in serum had HBV-DNA as well and that some patients had HBV-DNA in serum in the absence of HBsAg.
HBsAg, HBeAg, DNA polymerase, acute hepatitis B, chronic hepatitis B, hybridization
INTRODUCTION The polymerase chain reaction (PCR) technique is a method for amplifying nucleic acid by repeated cycles of hightemperature denaturation, oligonucleotide primer hybridization, and polymerase extension. Previous investigations with this technique have shown amplification of > 105-fold over the input quantity of nucleic acid ( 1 ) . In the past, two methods were available for detecting the presence of hepatitis B virus (HBV) DNA in serum: the endogenous DNA polymerase reaction (2) and dot- or slot-blot hybridization analysis (3-8). Of the two, slot-blot hybridization was demonstrated to be more sensitive; it can detect HBV- DNA in amounts as low as approximately 0.1 pg, corresponding to approximately 3 x lo4 virus genomes (3,5,7). PCR amplification has been used for detecting serum HBVDNA, and the sensitivity of the PCR assay was compared with that of slot-blot hybridization (9-1 1). Also, a rapid and sensitive PCR method for the detection of serum HBV-DNA was developed ( 12).
length ( 1 3). Several factors related to the selection of primer pairs for HBV-DNA (Fig. 1 ) detection were summarized (14): (a) primers specific for the single-stranded (primer pair 6 and 7) or double-stranded (primer pair 1 and 3) regions of the HBV genome are equally effective in amplifying serum HBVDNA; although it is possible that the single-stranded regions of the HBV genome are not amplified optimally during the first round of PCR, there is no significant effect found at the end of 30 cycles of amplification; (b) maximum application is seen using primers separated by no more than 500 nucleotides (e.g., primer pairs 1 and 2, 1 and 3, 6 and 7); and (c) primers with up to 14%mismatch (3 on the 21 nucleotides or primer 21-3) are as effective as the perfectly paired primer (primer 21) at amplifying HBV-DNA in a PCR assay using annealing temperatures of 36°C or 60°C. Since HBV genome sequences rarely vary by more than 10% worldwide, it is likely that primer pairs complementary to most regions of the virus genome should be appropriate for use in PCR assays.
SELECTION OF PRIMERS Systematic evaluation of the characteristics of optimal primer pairs is necessary for efficient PCR amplification of HBV-DNA, which is a circular molecule approximately 3,200 nucleotides long with a single-stranded region of variable 0 1990 Wiley-Liss, Inc.
Received May 14, 1990; accepted May 26, 1990 Address reprint requests to Shuichi Kaneko, M.D., First Department of Internal Medicine, Kanazawa University, Takara-Machi 13- 1, Kanazawa 920, Japan.
480
Kaneko et al. RAPID AND SENSITIVE PCR METHOD
5
Contamination with small amounts of DNA represents a considerable problem because of the sensitivity of PCR analysis. To eliminate sources of contamination, reagents were aliquotted and reaction mixtures were prepared with the use of disposable syringes and pipets. All reagents were assayed for the presence of HBV-DNA. However, these precautions are sometimes not enough to eliminate contamination; therefore, a procedure that minimizes false-positive results is - v necessary. Also, although PCR-SBH offers a tremendous increase in the ability to detect serum HBV-DNA, it is timeconsuming, involves several critical steps, and utilizes a radiolabeled probe that must be prepared frequently. A senc sitive and rapid PCR method that reduces the possibility of contaminating samples with exogenous HBV-DNA was developed ( 12 ) . In the NaOH extraction method, a 10-kl aliquot of serum was pipetted into a 0.5-ml microcentrifuge tube and incubated in 0.1 M NaOH at 37°C for 60 min. The solution was neutralized with HCl and amplified by PCR. The NaOH extraction method was either as effective as or slightly more effective than the phenol chloroform extraction method for Fig. 1. Map of the HBV genome and primers. The circular, partially release of HBV-DNA from serum particles for amplification double-stranded genome of HBV is shown with primers 1-8,21,21-3, and in the PCR assay (Fig. 2 ) . The HBV-DNA produced in a PCR 21-6. The position and direction of primers are shown on the map by arrows. Primer pairs separated by 160 base pair (bp) (primers 1 and 2), 270 bp (primers reaction mixture could be reamplified if the second primer I and 3), 553 bp (primers I and 4), 1,135 bp (primers 1 and 5), 2,037 bp pairs, internal to the original pair, were used. When an am(primers 1 and 7), and 2,623 bp (primers 1 and 8) were used to amplify equiv- plified reaction mixture was reamplified with internal primalent amounts of recombinant HBV-DNA. Primer pairs specific to single- ers, a DNA band of expected size was detected in samples stranded (primers 6 and 7) and double-stranded(primers 1 and 3) regions were containing more than lo-' pg of HBV-DNA before the first used to amplify serumHBV-DNA. Primers consisting of 21 nucleotides with no template mismatch (primer 21) or with three (primer 21-3) or six (21-6) PCR amplification reaction (PCR-PCR technique). Thus, nucleotides mismatched were used in a PCR reaction with primer 3 to detect equivalent amounts of recombinant HBV-DNA of known nucleotide sequence.
1 2 3 4 5 6 7 8 9
ESTABLISHINGTHE SENSITIVITY OF THE PCR TECHNIQUE Slot- or dot-blot hybridization analysis can detect
