The presence of hepatitis B virus ( HBV ) Deoxyribonucleic acid in serum is a dependable marker of viral reproduction and infectivity, and PCR, one of the molecular biological science techniques, is going the preferable method for its sensing. Fifty hepatitis B surface antigen ( HBsAg ) -positive serum samples were collected and used for DNA extraction utilizing three methods, including QIAamp MinElute Virus Spin Kit, DNPTM Kit and phenol-chloroform extraction process. Nested-PCR was carried out in order to find the presence of PCR inhibitors, and to compare three different methods of HBV DNA extraction from serum samples. Forty three out of 50 ( 86 % ) samples extracted by QIAamp MinElute Virus Spin Kit were positive for PCR. The consequence was 30 one out of 50 ( 62 % ) samples for DNPTM Kit and twenty out of 50 ( 40 % ) for phenol-chloroform extraction process. Our consequences 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.
Hepatitis B virus ( HBV ) , a 3.2 kilobit Orthohepadnavirus [ 1 ] , is a well-known agent of ague and chronic hepatitis, with an estimated 350 million chronic bearers around the universe [ 2, 3 ] . Efficient extraction of nucleic acids from clinical samples is extremely deciding for the dependability and public presentation of any molecular diagnostic check. Furthermore many extraction methodes are labour-intensive and time-consuming [ 4 ] . The presence of HBV DNA in serum is a dependable marker of genomic viral reproduction and infectivity [ 5 ] , and molecular biological science techniques show a powerful tool for nucleic acid analysis that enabling the sensing of few genomic transcript and are widely used for diagnostic and research intents [ 6 ] . The HBV DNA can be amplified by PCR and the PCR merchandises are visualized by ethidium bromide staining under ultraviolet visible radiation.
Because the efficaciousness of PCR elaboration may be affected by the presence of a figure of inhibitors of Taq polymerase enzyme in the serum, such as EDTA [ 7 ] , phenol [ 8 ] , polyamines [ 8 ] , polysaccharides [ 10 ] and Ca alginate [ 11 ] , DNA extraction is required before elaboration [ 5,12 ] .
Deoxyribonucleic acid 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 Deoxyribonucleic acid is of import for carry oning PCR and sequencing reactions [ 13 ] . Therefore the purpose of this survey was comparing of three different methods, including QIAamp MinElute Virus Spin Kit, DNPTM Kit and phenol-chloroform extraction process.
MATERIALS AND METHODS
Fifty hepatitis B surface antigen ( HBsAg ) – positive samples were collected from Persian Blood Transfusion Organization in Kermanshah state and were stored at -70 & A ; deg ; C until DNA extraction.
Deoxyribonucleic acid Extraction:
( A ) : Phenol-chloroform extraction process: a sum 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 assorted with an equal volume of trichloromethane: isoamylalcohol ( 24:1 ) . Then was precipitated with 3M Na ethanoate pH 5.2 and 96 % ethyl alcohol and incubated at -20 & A ; deg ; C for nightlong. After centrifugation for 15 min at 4 & A ; deg ; C at 16000g, the pellet was washed with 70 % cold ethyl alcohol and centrifuged at 16000g at 4 & A ; deg ; C. The pellet was air-dried and resuspended in 20µl double-distilled H2O ( dd H2O ) and so stored at -20 & A ; deg ; C until utilizing.
( B ) : QIAamp MinElute Virus Spin Kit ( QIAGEN, Germany ) : the extraction process was performed harmonizing to the maker ‘s instructions. 200µl buffer AL was added into the tubing including 200µl serum and 25µl QIAGEN peptidase and incubated at 56 & A ; deg ; C for 15 min in a warming block. 250µl of absolute ethyl alcohol was added and incubated at room temperature for 5 min. All of the lysate from the old measure briefly was applied onto a specific column and centrifuged at 6000g for 1 min. 500µl of buffer AW1 was added. After centrifugating at 6000g for 1 min, 500µl buffer AW2 was added and centrifuged at 6000g for 1 min. 500µl of absolute ethyl alcohol was so added and centrifuged at 6000g for 1 min. The column was dried at 56 & A ; deg ; C for 3 min. In ulterior measure, 100µl buffer AVE was added and centrifuged at 20000g for 1 min and so stored at -20 & A ; deg ; C until utilizing.
( C ) : DNPTM Kit ( CinnaGen Inc. ) : DNPTM Kit was another method for nucleic acerb extraction, purification and remotion of elaboration inhibitors that was used harmonizing to the maker ‘s instructions. After adding 5µl peptidase to 100µl serum, it was assorted with 400µl lysis solution and vortexed 20 sec. 300µl of precipitation solution was added, placed in -20 & A ; deg ; C for 20 min and centrifuged at 12000g for 10 min. 1ml rinsing buffer was so added to pellet and blend gently and so centrifuged at 12000g for 5 min. The pellet was dried at 65 & A ; deg ; C for 5 min, suspended in 50µl of dissolver buffer, shacked and placed at 65 & A ; deg ; C for 5 min. The mixture was centrifuged. Supernatant contained purified DNA.
for rating of three DNA extraction methods that present in this survey, nested PCR was done utilizing specific primers for HBV s cistron sequences harmonizing to antecedently described method by Zeng et Al. [ 14 ] .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 & A ; deg ; C, following of denaturation at 94 & A ; deg ; C for 45 sec, tempering for 60 sec at 55 & A ; deg ; C and extension at 72 & A ; deg ; C for 90 sec. Final extension was done at 72 & A ; deg ; 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 merchandises 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 usage of a lysis buffer and peptidase, and protocol A is based on Phenol-chloroform extraction followed by the ethanol precipitation. The Deoxyribonucleic acid extracted by three different methods was amplified by the nested PCR to find the presence of inhibitors of the Taq polymerase which can suppress PCR elaboration. ( Figure 1 ) . All three methods of DNA extraction could non wholly extinguish 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 set. Whereas this consequence was 31 of 50 ( 62 % ) for samples that were extracted by DNPTM Kit, that show the intermediate efficiency and proficiency consequence for HBV DNA extraction from infected serum samples. The less efficient consequence was obtained by Phenol-chloroform extraction process, as merely 20 out of 50 ( 40 % ) samples that extracted by this method were positive for PCR and revealed the 585bp set at stained agarose gel under ultraviolet visible radiation.
1 2 3 4 5 6 7 8 9 MTable 1. Primer sequences used for HBV genotyping in this survey
Zeng et Al. [ 14 ] By alteration in 4th nucleotide, from A to T
Sense, nt 2820-2837
Zeng et Al. [ 14 ]
Antisense, nt 842-821
Zeng et Al. [ 14 ]
Sense, nt 203-221
Zeng et Al. [ 14 ] By alteration in 14th 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 merchandises of Deoxyribonucleic acid extracted by protocol A, Lines 4-6: PCR merchandises of Deoxyribonucleic acid extracted by protocol B, Lines 7-9: PCR merchandises of Deoxyribonucleic acid extracted by protocol C. Line M: Marker 100bp plus DNA ladder.
A suited method for extraction of nucleic acids should be efficient, sensitive, rapid and simple. Furthermore the method should give pure nucleic acid free from any PCR elaboration inhibitor. Therewith the process should forestall any cross-contamination among samples that are extracted by a synchronous processing [ 6 ] .
The primary purpose of this survey was to measure and compare of three DNA extraction methods for isolation of HBV genome from infected serum samples. Therefore the genomic merchandises that were extracted by all of the three methods for DNA extraction from HBV infected serum samples were entered into PCR elaboration. PCR is presently a common and widespread tool for measuring the efficiency, proficiency and sensitiveness of different nucleic acid extraction processs. However, the public presentation of PCR in measuring assorted extraction method is dependent on riddance of PCR inhibitors Hence, utilizing specific primers for s cistron of HBV, PCR was performed on all merchandises that were extracted by three different methods. We could successfully magnify a 585bp fragment of HBV s cistron to compare and measure three different DNA extraction end product.
During rating of the three different DNA extraction methods for pull outing DNA virus of septic serum samples, PCR was carried out with 2.5 ?l extracted DNA for a 25 ?l entire volume reaction.
Assume the first measure of all DNA extraction processs apply a lysis [ 15-17 ] . In order to liberate nucleic acids that can so be purified, this measure was done in all of the three different HBV DNA extraction from serum samples. The working rule of QIAamp MinElute Virus Spin Kit and DNPTM Kit was efficient use of peptidase and lysis buffer for lysing the virions but peptidase was non used in Phenol-chloroform extraction process. The detergents were used to solubilize any cell constituents in all three DNA extraction processs that were described in this survey.
There are several studies 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 civilization negative intraocular aspirates [ 18 ] .
The NucliSens Extractor is an machine-controlled GuSCN silicabased nucleic acerb extraction method which was able to expeditiously insulate HBV DNA from pretreated plasma and serum samples, leting sensitive sensing of HBV by a noncommercial PCR method [ 4 ] .
Harmonizing to the consequences obtained in this survey, 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 velocity of the procedure in less than 1 hr. This method could cancel taints, PCR inhibitors and repressive factors of Taq polymerase activity because 86 % of extracted samples that were extracted by this method were positive for PCR elaboration and showed the 585bp set. Since this kit is done by utilizing QIAamp MinElute columns, there is no sample-to sample cross-contamination. Other two protocols incorporating DNPTM Kit and Phenol-chloroform extraction process were acted weaker than QIAamp MinElute Virus Spin Kit as merely 62 % of samples extracted by utilizing DNPTM Kit and 40 % of samples extracted by utilizing phenol-chloroform extraction process were positive for PCR. This consequence is presumptively due to the presence of more taint and PCR- inhibitors and uncomplete omission of inhibitors of Taq polymerase that found in the serum.
However DNPTM Kit requires a little sum of serum, merely 100µl, and can be used in instances where a little sum of serum sample is available. Phenol-chloroform extraction method was non a suited and effectual process for HBV DNA extraction from HBsAg-positive serum samples as described above. This method is really time-consuming and there is a hazard of sample-to-sample cross-contamination.
In term of cost, QIAamp MinElute Virus Spin Kit was the most expensive method and can be an effectual option when cost is a confining factor.
In decision, the consequences of this survey indicate that each of the probe method for extraction of nucleic acid has net income and harmful familiarities, and we should study advantages and disadvantages of it when is selected for the probe.
We thank Dr. Mozaffar Sharifi and Mrs. Sedigheh Arab for their counsels