The presence of hepatitis B virus (HBV) DNA in serum is a reliable marker of viral replication and infectivity, and PCR, one of the molecular biology techniques, is becoming the preferred method for its detection. Fifty hepatitis B surface antigen (HBsAg)-positive serum samples were collected and used for DNA extraction using three methods, including QIAamp MinElute Virus Spin Kit, DNPTM Kit and phenol-chloroform extraction procedure. Nested-PCR was carried out in order to determine the presence of PCR inhibitors, and to compare three different methods of HBV DNA extraction from serum samples. Forty three out of fifty (86%) samples extracted by QIAamp MinElute Virus Spin Kit were positive for PCR. The result was thirty one out of fifty (62%) samples for DNPTM Kit and twenty out of fifty (40%) for phenol-chloroform extraction procedure. Our results reveal that QIAamp MinElute Virus Spin Kit is the most efficient method for HBV DNA extraction of serum samples, and it's PCR inhibitors is less than two other methods.
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Hepatitis B virus (HBV), a 3.2 kb Orthohepadnavirus , is a well-known agent of acute and chronic hepatitis, with an estimated 350 million chronic carriers around the world [2, 3]. Efficient extraction of nucleic acids from clinical samples is highly determinant for the reliability and performance of any molecular diagnostic assay. Moreover many extraction methodes are labor-intensive and time-consuming . The presence of HBV DNA in serum is a reliable marker of genomic viral replication and infectivity , and molecular biology techniques show a powerful tool for nucleic acid analysis that enabling the detection of few genomic copy and are widely used for diagnostic and research purposes . The HBV DNA can be amplified by PCR and the PCR products are visualized by ethidium bromide staining under ultraviolet light.
Because the efficacy of PCR amplification may be affected by the presence of a number of inhibitors of Taq polymerase enzyme in the serum, such as EDTA , phenol , polyamines , polysaccharides  and calcium alginate , DNA extraction is required before amplification [5,12].
DNA extraction can be done by many methods that some of them are standard methods. An efficient method of DNA extraction that yields pure and high-quality DNA is important for conducting PCR and sequencing reactions . Therefore the aim of this study was comparison of three different methods, including QIAamp MinElute Virus Spin Kit, DNPTM Kit and phenol-chloroform extraction procedure.
MATERIALS AND METHODS
Fifty hepatitis B surface antigen (HBsAg)- positive samples were collected from Iranian Blood Transfusion Organization in Kermanshah province and were stored at -70°C until DNA extraction.
(A): Phenol-chloroform extraction procedure: a total of 200µl of each sample was added to 200µl of lysis buffer ( 25mM EDTA, 200mM Tris-HCl pH 7.5, 250mM NaCl, 1%SDS ) and was included at room temperature for 60 min. 400µl equilibrated phenol pH 7.8 was added, decanted and incubated for 10 min at room temperature. Tubes were centrifuged at 12000g for 5 min. Then mixed with an adequate volume of chloroform:isoamylalcohol (24:1). Then was precipitated with 3M sodium acetate pH 5.2 and 96% ethanol and incubated at -20°C for overnight. After centrifugation for 15 min at 4°C at 16000g, the pellet was washed with 70% cold ethanol and centrifuged at 16000g at 4°C. The pellet was air-dried and resuspended in 20µl double-distilled H2O (dd H2O) and then stored at -20°C until using.
(B): QIAamp MinElute Virus Spin Kit (QIAGEN, Germany): the extraction procedure was performed according to the manufacturer's instructions. 200µl buffer AL was added into the tube including 200µl serum and 25µl QIAGEN protease and incubated at 56°C for 15 min in a heating block. 250µl of absolute ethanol was added and incubated at room temperature for 5 min. All of the lysate from the previous step briefly was applied onto a specific column and centrifuged at 6000g for 1 min. 500µl of buffer AW1 was added. After centrifuging at 6000g for 1 min, 500µl buffer AW2 was added and centrifuged at 6000g for 1 min. 500µl of absolute ethanol was then added and centrifuged at 6000g for 1 min. The column was dried at 56°C for 3 min. In later step, 100µl buffer AVE was added and centrifuged at 20000g for 1 min and then stored at -20°C until using.
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(C): DNPTM Kit (CinnaGen Inc.): DNPTM Kit was another method for nucleic acid extraction, purification and removal of amplification inhibitors that was used according to the manufacturer's instructions. After adding 5µl protease to 100µl serum, it was mixed with 400µl lysis solution and vortexed 20 sec. 300µl of precipitation solution was added, placed in -20°C for 20 min and centrifuged at 12000g for 10 min. 1ml washing buffer was then added to pellet and mixed gently and then centrifuged at 12000g for 5 min. The pellet was dried at 65°C for 5 min, suspended in 50µl of solvent buffer, shacked and placed at 65°C for 5 min. The mixture was centrifuged. Supernatant contained purified DNA.
for evaluation of three DNA extraction methods that present in this study, nested PCR was done using specific primers for HBV s gene sequences according to previously described method by Zeng et al. .by some modifications.The sequences of the primers are shown in Table1. Nested PCR, in first- and second-round PCR, was performed for 3 min at 94°C, following of denaturation at 94°C for 45 sec, annealing for 60 sec at 55°C and extension at 72°C for 90 sec. Final extension was done at 72°C for 6 min. PCR solution contained 2.5µl of extracted DNA, 0.5µl dNTP mix, 2.5µl 10x Taq polymerase buffer, 0.75µl MgCl2, 0.2µl Taq polymerase and 1µl of each primer (10 pmol), PrsS3 and S1R in first-round PCR and YS1 and YS3 in second-round PCR. PCR products were detected in 1% ethidium bromide-stained agarose gel.
Protocol B (QIAamp MinElute Virus Spin Kit) and protocol C (DNPTM Kit) are based on the use of a lysis buffer and protease, and protocol A is based on Phenol-chloroform extraction followed by the ethanol precipitation. The DNA extracted by three different methods was amplified by the nested PCR to determine the presence of inhibitors of the Taq polymerase which can inhibit PCR amplification. (Figure 1). All three methods of DNA extraction could not entirely eliminate the PCR inhibitors in all serum samples.
43 of 50 (86%) samples that were extracted by QIAamp MinElute Virus Spin Kit were positive for PCR and revealed 585bp band. Whereas this result was 31 of 50 (62%) for samples that were extracted by DNPTM Kit, that show the intermediate efficiency and proficiency result for HBV DNA extraction from infected serum samples. The less efficient result was obtained by Phenol-chloroform extraction procedure, as only 20 out of 50 (40%) samples that extracted by this method were positive for PCR and revealed the 585bp band at stained agarose gel under ultraviolet light.
1 2 3 4 5 6 7 8 9 MTable 1. Primer sequences used for HBV genotyping in this study
Zeng et al.  By modification in fourth nucleotide, from A to T
Sense, nt 2820-2837
Zeng et al.
Antisense, nt 842-821
Zeng et al.
Sense, nt 203-221
Zeng et al.  By modification in fourteenth nucleotide, from T to C
Antisense, nt 787-767
Figure 1. Detection of HBV DNA by nested PCR in serum samples following extraction by different methods. Lines 1-3: PCR products of DNA extracted by protocol A, Lines 4-6: PCR products of DNA extracted by protocol B, Lines 7-9: PCR products of DNA extracted by protocol C. Line M: Marker 100bp plus DNA ladder.
A suitable method for extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover the method should yield pure nucleic acid free from any PCR amplification inhibitor. Therewith the procedure should prevent any cross-contamination among samples that are extracted by a synchronic processing .
The primary aim of this study was to evaluate and compare of three DNA extraction methods for isolation of HBV genome from infected serum samples. Therefore the genomic products that were extracted by all of the three methods for DNA extraction from HBV infected serum samples were entered into PCR amplification. PCR is currently a common and widespread tool for evaluating the efficiency, proficiency and sensitivity of different nucleic acid extraction procedures. However, the performance of PCR in evaluating various extraction method is dependent on elimination of PCR inhibitors Hence, using specific primers for s gene of HBV, PCR was performed on all products that were extracted by three different methods. We could successfully amplify a 585bp fragment of HBV s gene to compare and evaluate three different DNA extraction output.
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During evaluation of the three different DNA extraction methods for extracting DNA virus of infected serum samples, PCR was carried out with 2.5 μl extracted DNA for a 25 μl total volume reaction.
Assume the first step of all DNA extraction procedures apply a lysis [15-17]. In order to free nucleic acids that can then be purified, this step was done in all of the three different HBV DNA extraction from serum samples. The working principle of QIAamp MinElute Virus Spin Kit and DNPTM Kit was efficient usage of protease and lysis buffer for lysing the virions but protease was not used in Phenol-chloroform extraction procedure. The detergents were used to solubilize any cell components in all three DNA extraction procedures that were described in this study.
There are several reports about HBV genomic extraction from infected serum samples evaluated and compared by PCR technique [4, 5, 6]. PCR has Other applications such as HSV 1 and 2 serotyping in culture negative intraocular aspirates .
The NucliSens Extractor is an automated GuSCN silicabased nucleic acid extraction method which was able to efficiently isolate HBV DNA from pretreated plasma and serum samples, allowing sensitive detection of HBV by a noncommercial PCR method .
According to the results obtained in this study, QIAamp MinElute Virus Spin Kit was more efficient method for HBV DNA extraction from infected serum samples that were HBsAg-positive. Therewith this method is less time-consuming and increase the speed of the process in less than 1 hour. This method could delete contaminations, PCR inhibitors and inhibitory factors of Taq polymerase activity because 86% of extracted samples that were extracted by this method were positive for PCR amplification and showed the 585bp band. Since this kit is done by using QIAamp MinElute columns, there is no sample-to sample cross-contamination. Other two protocols containing DNPTM Kit and Phenol-chloroform extraction procedure were acted weaker than QIAamp MinElute Virus Spin Kit as only 62% of samples extracted by using DNPTM Kit and 40% of samples extracted by using phenol-chloroform extraction procedure were positive for PCR. This result is presumably due to the presence of more contamination and PCR- inhibitors and incomplete deletion of inhibitors of Taq polymerase that found in the serum.
Nevertheless DNPTM Kit requires a small amount of serum, only 100µl, and can be used in cases where a small amount of serum sample is available. Phenol-chloroform extraction method was not a suitable and effective procedure for HBV DNA extraction from HBsAg-positive serum samples as described above. This method is very time-consuming and there is a risk of sample-to-sample cross-contamination.
In term of cost, QIAamp MinElute Virus Spin Kit was the most expensive method and can be an effective option when cost is a limiting factor.
In conclusion, the results of this study indicate that each of the investigation method for extraction of nucleic acid has profit and harmful intimacies, and we should survey advantages and disadvantages of it when is selected for the investigation.
We thank Dr. Mozaffar Sharifi and Mrs. Sedigheh Arab for their guidances