INTRODUCTION  

Cancer of the cervix is an important public  health  problem  worldwide  and  is  the second  most  common  cancer  among women  (Woodman  et  al.  2007).  In  Malaysia,  it  is  the  second  most  common cancer among women in all major ethnic groups  (Ministry  of  Health  Malaysia 2003). Human papilloma virus (HPV) has been  identified  as  the most  important  viral group associated with benign and ma- lignant neoplasia in humans; HPV DNA is estimated to be present in over 99.7% of these  cancers  (Walboomers  et  al.  1999; Lockwood  &  Mcintyre  2009).  Using  real time PCR method, Panu et al. (2002) and Bourgo  et  al.  (2008)  showed  the  presence of integrated HPV type 16  in cervical  Intraepithelial  neoplasia  (CIN).  Inte- gration  of  viral  genome  into  the  nucleus has been proposed as the mechanism for activation,  proliferation  and  progression from  non-invasive  lesions  to cervical cancers (Mitsuo et al. 1999; Fan & Chen 2004;  Lockwood  & Mcintyre  2009).  The Hybrid  Capture  (HC)  assay,  the  current diagnostic test for the screening of cervical  cancer,  characterizes  HPV  infections as  low  or  medium/high  risk  but fails  to identify  HPV  infections  by  type  (Sonnex 1998; Monica et al. 2002 & Seaman et al. 2010).  Real  time  PCR  is  an efficient technique  that  objectively  detects  and quantifies DNA target sequence in a period of two or three hours, whereas classical  PCR  needs  at  least  four  hours  to detect the same sequence without quantifying it (Bettini et al. 2008).  

Recently,  two  vaccines  were made available  for  protection  of  infection  by some  HPV types,  Gardasil,  marketed  by Merck  and Cervarix,  marketed  by GlaxoSmithKline  (Moscicki  2008). Both protect  against initial infection  with  HPV types 16 and 18, which cause most of the HPV  associated  cancers.  Gardasil also protects  against HPV  types  6 and 11, which cause 90% of genital warts (Asif et al. 2006; Kahn 2009; WHO 2009 & Schil- ler et al. 2010). The vaccines provide little benefit to women who have already been infected with HPV types 16 and 18, which include most sexually active females. The World  Health  Organization (WHO  2009) has  clear  guidelines on HPV  vaccination which  outlines appropriate,  cost-effective strategies for using HPV vaccines in public sector programs.  

Cervical cancer is caused by a virus; it can be prevented in two ways: (i) by preventing  HPV  infection  by  using  a poten- tial  vaccine  and  (ii)  by  screening  for  the presence  of  cervical  HPV  infection  and managing  abnormal  cytologies  or  pre-cancerous  lesions  before  cancer  develop.  In Malaysia  however,  the  lack  of an  adequate  screening  programme  and awareness are the major factors contrib- uting  to the  high  incidence  of  cervical cancer.  The  main  purpose  of  this  study was to establish a simple and rapid technique  to  detect  high  risk  HPV  subtypes using  SyBrGreen  Real-Time  PCR.  This technique is advantageous since it eliminates  the  need  for  gel  electrophoresis and  ethidium  bromide  staining.  The  method  of  SyBr-Green  Real-Time  PCR  can be used as a screening approach to controlling cervical cancer. 

MATERIALS AND METHODS

Samples 

Genomic  DNA  from  57  HPV  positive samples  for  HPV  16  and  HPV  18  identified  from  our  Type  Specific-PCR  (TS- PCR)  was used for this study. The sam- ples  include 37 CIN I, 12  CIN II, 15 CIN III  and  3  squamous  cell  carcinoma (SCC).  CaSki  and  HeLa  cell  lines  were used as positive controls for HPV 16 and HPV  18,  respectively.  For  negative  control, blood DNA from normal women was chosen.  DNA  extraction  was  done  using the  QIAamp  Tissue  kit  from  QIAGEN (Germany).   

Cell culture 

The  vial  containing  cell-lines  was  removed  from  the  liquid  nitrogen  tank  and was  immediately  thawed  into  a  water bath at 37ºC. The content of the vial was transferred into a centrifuge tube and 10 ml  of  growth  medium  were  added.  The tube  was centrifuged  at 1000 rpm for 10 minutes.  Supernatant  was  removed  and cells were resuspended in 5 ml of growth medium,  later  transferred  to  a  culture flask and incubated in a 37ºC CO2  incubator. The medium was replaced the following  day.  Subculture  was  carried  out by  detaching  cells  using  tripsin.  One  ml of trypsinethylenediaminetetraacetic acid (EDTA)  solution  (Flowlab)  was  added  to the flask. The flasks were incubated in a CO2 incubator  for  5  minutes.  The  flask was  then  observed  under  the  inverted microscope  to  check  for  the  detachment of  cells  and  was  tapped  gently  to  optimize cell detachment.   

Real-Time PCR with SyBr Green 

Genomic  DNA  from  57 positive  samples for  HPV  16 and  HPV  18 were  used  to prepare  a triplicate  reaction  for  each sample  using  Real-Time  PCR.  Primers from  TS-PCR  (Walboomers  et al.  1999) were used as a primer pair for HPV type 16  and  18  in  the  SyBr  Green  Real-Time PCR.  Amplification  reactions  were  per- formed  with a volume  of 12.5µl  of  2X SyBrGreen  PCR  Master  Mix  (Applied Biosystems), a total of 300nM of reverse and forward primers and 20ng of sample DNA in a 25µl final volume. The protocol was set up at 95oC for 10 minutes to activate  the Taq  gold,  40 cycles  at  95oC  for 15  sec  and  60oC  for  15 sec,  consecu- tively.  Each  reaction  was  accompanied by a No Template Control (NTC) in which the template DNA was replaced by water. Amplification  was  performed  in  a 248- well PCR plate covered with optical caps in  the ABI  7700HT-SDS  real-time  instrument (Applied Biosystem).

RESULTS

Optimization of SyBr Green Reaction 

From our results using SyBr Green Real Time PCR, NTC was used as a negative control; CaSki and HeLa DNA were used as a positive control for HPV 16 and HPV 18  respectively.  Dissociation  curve  was generated  to  show  the  absence  of  non- specific  amplification.  The  dissociation curve  showed  only  a  single  peak  at higher  temperature  and  two  peaks  if  primer-dimer  occurred.  According  to  Lamarcq et al. (2002), Cycle threshold (Ct) value  was  considered  significant  when  it was  at  least  four  cycles  lower  than  Ct value of the corresponding NTC. Another study by Szuhai et al. (2001), reported that a Ct value is significant when it was lower than 40 cycle’s number. Figure (1a) shows the amplification plot of HPV 16 with negative control NTC, Ct value at 20 cycles. Primer-dimer of NTC was observed with a Ct value at 25 cycles. The Ct value of HPV 16 was significant because the differences between HPV 16 and NTC were five cycles. Figure (1b) shows dissociation curve of HPV 16. Two peaks were observed at 80.7ºC for melt-ing temperature Tm of specific HPV 16 and at 74ºC for a NTC. Figure (2a) shows the amplification plot of HPV 18 with NTC and Ct value for HPV 18 was 21 cycles. Primer-dimer of NTC was ob-served with a Ct value at 28 cycles. The Ct value of HPV 18 was significant because the differences between HPV 18 and NTC were seven cycles. Figure (2b) shows dissociation curve of HPV 18. Two peaks were observed at 79.3ºC for melting temperature T_m of specific HPV 18 and at 70ºC for a NTC.

Identification of multiple infections of HPV types

Results from our previous TS-PCR, showed six samples were positive for multiple infections by HPV 16 and HPV 18. Figure 3 showed a dissociation curve of HPV 16 and HPV 18. Four peaks were displayed; one peak for HPV 16, one peak for HPV 18 and two peaks for NTC. Melting temperature T_m for HPV 16 was 80.7°C and for NTC was 74°C, whereas for HPV 18, melting temperature Tm was 79.3°C and for NTC was 70°C. SyBr Green Real Time PCR method showed the Tm for HPV 16 and HPV 18 were different although the PCR products using TS-PCR for HPV 16 and HPV 18 produced a single band as a 100 bp. The dissociation curves using SyBr Green Real Time PCR method showed that the T_m for HPV 16 was 80.6°C whereas Tm for HPV 18 was 79.4°C. Studies by Szu-hai et al. (2001) and Kleinle et al. (2002) suggested the T_m of an amplification product is based on amplicon size and GC content; fragments of different length and G+C content can be distinguished by melting curve analysis. Our study found that the T_m  of HPV 16 was higher than T_m of HPV 18.

Identification of multiple infections of HPV types

Results from our previous TS-PCR, showed six samples were positive for multiple infections by HPV 16 and HPV 18. Figure 3 showed a dissociation curve of HPV 16 and HPV 18. Four peaks were displayed; one peak for HPV 16, one peak for HPV 18 and two peaks for NTC. Melting temperature T_m for HPV 16 was 80.7°C and for NTC was 74°C, whereas for HPV 18, melting temperature T_m was 79.3°C and for NTC was 70°C. SyBr Green Real Time PCR method showed the T_m for HPV 16 and HPV 18 were different although the PCR products using TS-PCR for HPV 16 and HPV 18 produced a single band as a 100 bp. The dissociation curves using SyBr Green Real Time PCR method showed that the T_m for HPV 16 was 80.6°C whereas T_m for HPV 18 was 79.4°C. Studies by Szu-hai et al. (2001) and Kleinle et al. (2002) suggested the T_m of an amplification product is based on amplicon size and GC content; fragments of different length and G+C content can be distinguished by melting curve analysis. Our study found that the T_m of HPV 16 was higher than T_m   of HPV 18.

Table 1 showed the C_t value of HPV 16 and HPV 18 were below 40 cycles and also differs from NTC by six cycles so that the C_t values were significant based on studies by Szuhai et al. (2001) and Lamarcq et al. (2002). This table also showed the differences of T_m between specific product of HPV 16 and NTC were 6.7°C whereas the differences of T_m between specific product of HPV 18 and NTC were 9.3°C. Study by Kleinle et al. (2002), suggested the T_mof the gene specific product differs from primer-dimers by at least 10°C, the peak of the gene-specific product did not overlap with the primer-dimer peak and thus the accuracy of the quantification was validated.

DISCUSSION

Amplification plot of HPV 16 and HPV 18

In this study, minimum primers for HPV 16 and 18 were 300:300nM for forward and reverse primers and DNA concentration at 100 ηg. All graphs from Figure 1-3 showed C _t value for HPV 16 was 20±1 cycles and dissociation curve of HPV 16 with melting curve T_m was 80.7±0.2ºC whereas melting curve T_m for NTC was 73.5±0.2ºC. Amplification plot of HPV 18 showed presence of two curves with one specific for HPV 18 and another one for NTC. C_t value for HPV 18 was 21±1cycles and C_t value for NTC was 28±1 cycles. Table 1 showed the C_t value of HPV 16 and 18 were significant because, the difference between specific product and NTC was five and seven cycles (Kleinle 2002). For melting temperature, T_m shows the differences of T_m between HPV 16 and NTC were below 10°C such as 7.2°C whereas for HPV 18 the differences between specific HPV 18 and NTC were 9.4°C. Kleinle et al. (2002), suggested the T_m of the gene specific product differs from primer-dimers or NTC by at least 10°C, the peak of the gene-specific product did not overlap with the primer-dimer peak and thus the accuracy of the quantification was validated. However, this study found the differences of T_m for HPV 16 and HPV 18 were below 10°C, this phenomena happened because the amplicon size of HPV 16 and 18 were 100bp and the size of NTC range from 50-60bp. The close-up of amplicon size between specific product and NTC will affect the T_m. Although the amplicon size between spe-cific products and NTC was very close, the graph shows gene-specific product of HPV 16 and HPV 18 did not overlap with the NTC peak so the accuracy of the qualification was validated.

Identification of multiple infections of HPV types

From our previous sequencing results of PCR products of E7 gene for HPV 16 (RID 1055387141-08795-2060) and HPV 18 (RID 1056435350-019738-16228) we observed that, GC content for HPV 16 was higher than the GC content of HPV 18, and the PCR product of HPV 16 had high T_m compared to T_m of HPV 18. The differences in melting temperature can be used as a method to identify and diffe-rentiate multiple infections between HPV16 and HPV 18. Kleinle et al. (2002) and Lamarcq et al. (2002) reported that multiple specific products can be sepa-rated from each other due to different T_m in most cases. This method is advantageous since it eliminates the need for gel electrophoresis. This finding shows, SyBr Green can be used to differentiate and identify HPV types. Table 1 shows a summary of the C_t values and T_m for HPV 16 and HPV 18. The C_t value of HPV 16 and HPV 18 were below 40 cycles and also differ from NTC by six cycles, indicating that the C_t values were significant. The findings were supported by studies by Szuhai et al. (2001) and Lamarcq et al. (2002). Table 1 also showed the differences of T_mbetween HPV 16 and HPV 18 with the NTCs being 6.7°C and 9.3°C respectively. The peak of the gene-specific product did not overlap with the primer-dimer peak and thus the accuracy of the quantification was validated. This SyBr Green Real- Time PCR method offers the possibility of substantially increasing the throughput of sample analysis, of analyzing different samples for the presence of any of these gene mutations and/or determining the individual gene mutation pattern. In terms of the rapidity, flexibility and the subsequent economic advantages, this approach is suitable for use in clinical and routine laboratory applications (Cubie et al. 2001; Szuhai et al. 2001; Kleinle et al. 2002; Villa et al. 2006).

CONCLUSION

Using SyBr Green Real-Time PCR technique, minimum forward and reverse primers for each HPV type 16 and 18 were required. DNA concentration for each samples were fixed at 100ng. SyBr Green Real-Time PCR method has the advantage of it being able to be used directly to detect and identify the HPV types based on amplification plot and melting temperature graphs. This SyBr Green Real-Time PCR method has the potential to be used as a method for de-tection and identification of HPV types in CINs and cervical cancer patients in Malaysia. In addition it has the advantage of detecting multiple infections of HPVs which adds to its economic value.

ACKNOWLEDGEMENTS

The study was supported by a postgraduate grant from Institute Bioscience (IRPA-5495304), Universiti Putra Malaysia. The authors wish to thank the Director General of Health, Malaysia for permission to publish this scientific paper. We also like to thank the Deputy Director General (Research & Technical Support, MOH) and the Director of IMR for their support.