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Contents
Chapter 1: Introduction 1
1-1. The introduction of human papillomavirus (HPV) 1
1-2. HPV epidemiology 2
1-3. Human papillomavirus vaccine 3
1-4. HPV diagnostic methods 4
1-5. High-resolution melting analysis (HRM) 5
1-6. Unlabeled oligoantisense (unlabeled probe) 6
1-7. Asymmetric HRM 7
Chapter 2: Methods 9
2-1. Clinical sample preparation 9
2-2. Specimen DNA quality check and cloning preparation 9
2-3. High-resolution melting analysis: asymmetric and symmetric PCR 10
2-4. Electrophoresis 11
2-5. Plasmid construction with TA-cloning 11
2-6. Plasmid DNA extraction and preservation 12
Chapter 3: Results 13
3-1. Basic condition monitoring by conventional PCR 13
3-2. Plasmid construction 17
3-3. Standard curve construction with high-resolution melting 18
3-4. Clinical test 22
Chapter 4: Discussion 24
4-1. Specific genotype analysis 24
4-1-1. HPV31 and 42 24
4-1-2. HPV33 26
4-1-3. HPV39 26
4-1-4. HPV45 28
4-1-5. HPV51 29
4-1-6. HPV56 29
4-1-7. HPV68 30
4-2. Summary 30
References 32
Tables and Figures 41
Figure 1. The SPF1-GP6+ PCR result with two annealing temperature tests. 41
Figure 2. 8 genotype amplified by FRG-1-GP6+. 42
Figure 3. Three primer pair tests with two magnesium ion concentrations. 43
Figure 4. New primer pairs tests. 45
Figure 5. PCR condition determinative tests. 47
Figure 6. Plasmid confirming and gradient tests. 49
Figure 7. The concentration factor in symmetric HRM. 50
Figure 8. Eight genotypes’ analysis in symmetric HRM. 52
Figure 9. The difference with HPV16-specific probe added. 53
Figure 10. HPV18 gradient test with HPV18-specific probe added. 55
Figure 11. Seven genotypes in asymmetric HRM. 57
Figure 12. Two magnesium ion concentration tests. 58
Supplements 60
Supplement-1. 13 HR-HPV DNA sequences. 60
Supplement-2. HPV31 and 42 sequence comparison 61
Supplement-3. HPV39 variants 62
Supplement-4. HPV45 variants. 63
Supplement-5. Clinical data. 64
Chapter 1: Introduction
1-1. The introduction of human papillomavirus (HPV)
Human papillomavirus (HPV) is a naked, non-enveloped double-strand DNA virus. It consists of two groups of genes, late genes (L) and early genes (E). The late genes, including L1 (major capsid protein) and L2 (minor capsid protein) construct the HPV viral particle. The early genes, including E1, E2, E4, E5, E6 and E7, regulate the HPV replication and proliferation (33, 44). In recent years, scientists have discovered more than 100 HPV genotypes (6). Among these HPV genotypes, more than 40 genotypes have been defined to infect the genital tract. Thirteen to fifteen HPV genotypes are called high-risk HPV (HR-HPV) which is determined by the FDA-approved approach, hybrid capture (HC) (9, 23, 29). Including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 (73 and 82 are counted in 15 HR-HPV) (8.10). The HR-HPV virus integrates into the host genome, especially in the basement membrane cells at neck of the female uterus, where it induces continuous epithelial cell inflammation and transformation, like low squamous intracellular lesion (LSIL), high squamous intracellular lesion (HSIL), and even transforms to cervical cancer (6). However, HR-HPV infection is a long-term progressive diseases; it can be reversed under appropriate therapy in early and middle stage (35).
Not only HR-HPV induces genital tract diseases, but also low-risk HPV (LR-HPV). LR-HPV will induce other common genital diseases; warts is seems to be the most common genital disease induced by LR-HPV, especially by HPV6 and HPV11. Based on the infection site, most warts cases are infected by sexual behavior. In other words, no matter HR-HPV infection or LR-HPV infection, most of them are called sexual transmission disease (STD).
1-2. HPV epidemiology
In recent years, scientists have determined 13 HPV genotypes as the high-risk HPV (HR-HPV) which represent the most dangerous HPV genotypes, according to an FDA-approved clinical method, hybrid capture (HC). However, the epidemiologic percentage of HR-HPV is not always constant in each country. Oppositely, while HPV16 and 18 are usually the top two leaders of clinical cases, the other 11 HR-HPV genotypes are various. For example, according to the cross-national research, the 13 HR-HPV represent over 95％ of cervical cancer cases including the single genotype and multiple genotype infection (26). In particular, HPV16 and HPV18 together of them comprise about 67.7％ (HPV16: 54.6％, HPV18: 11.0, HPV16 + 18 : 2.1％) of cervical cancer cases. In Taiwan, HPV 16 was detected in 63.8% of the cervical cancer cases, and HPV 18 was detected in 16.5% of cervical cancer cases (17, 18). Obviously, if we only compare the two major HR-HPV genotypes, the HPV epidemiology of HPV16 and 18 in Taiwan is as same as that worldwide. But there are some differences in Taiwan; they are HPV52 and 58. In fact, the epidemiology in south-east Asia (Taiwan, Hong Kong and Singapore) is huge different to the other countries. In worldwide epidemiology, HPV52 is detect in 2.2％ and HPV58 is detected in 2.0％ of clinical cases (26). In Taiwan, HPV52 is detected in 6.8％ and HPV58 is detected in 16.3％ (17, 18). And the clinical evidence shows that HPV52 and 58 infection cases are more than those of HPV16 and 18 in early stage (35). So it is easy to predict that HPV52 and 58 may be the highest risk HPV genotypes during the development of HPV vaccine in Taiwan. With this situation, the variation of HPV epidemiology will be a huge research topic with the development of HPV vaccine.
1-3. Human papillomavirus vaccine
During the 1990s, scientists provide a new idea that we may use multiple HPV recombined proteins which are produced from different HPV genotypes as HPV vaccine. However, there is no research group indicating that the multiple HPV recombined proteins succeed in HPV vaccination because scientists discover that the multiple recombined proteins can’t induce a properly and accurate immune reaction.
After the 2000s, one company, Merck firstly manufactured the L1 protein to construct a HPV viral-like particle (VLP) as the vaccine. Gardasil vaccine is a quadrivalent vaccine includes HPV6, 11, 16 and18. In theory, the vaccine produced by those companies like Merck and GSK could prevent a lot of patients from the menace of some HPV genotypes. It could prevent up to 70％ (HPV16 and 18) of cervical cancer cases and reduce the cost of cancer therapy and cervical screening programs (44). In other words, if women receive Merck HPV vaccine injection, they could be protected from some HPV genotypes, like HPV6, 11, 16 and 18. However, that may induce a giant change in HR-HPV epidemiology because the vaccination immunizes against a portion of HPV genotypes.
It’s expectable situation that the incidences of HPV 16 and 18 will reduce and other HR-HPV genotypes will take their place. But we don’t know which genotype will be the dominant genotype, despite the possibility of HP52 and 58 are higher than the other HR-HPV. However, it’s necessary to a build a monitoring mechanism to observe the variation of HR-HPV epidemiology.
1-4. HPV diagnostic methods
In recent years, there have been many commercial kits produced, like the Roche linear array (LA), Digene Hybrid Capture 2 (HC2), and KingCar DNA microarray (Genechip). Each commercial kit is produced depending on different designed background and experimental aim. Among these HPV genotyping systems, Hybrid Capture 2 (HC2) is the most well-known method for detecting HPV. The manufacturer, Digene Corporation had developed it into the second generation (HC2) as of 2009. HC2 is the FDA (Food and Drug Administration, American) approved method. It is unable to distinguish which genotype the specimen of patient is, but it contains the most powerful ability to detect the presence or absence of HPV infection. So far, HC2 is the first choice among methods for the doctor who wants to know if a patient is infected with the HPV or not. The basic principle of HC2 is based on the signal amplification of the enzyme immunoassay (EIA). HC2 uses two types of DNA/RNA specific Ab; type one is the beads coated with enzyme (ACP)-conjugated Ab, and type two Ab is coated on the reaction well, becoming the capture anchor. In addition to the DNA/RNA specific Ab, HC2 uses different genotype-specific RNA probes (13 HR-HPV specific probes) which can recognize the target sequence of HPV DNA to detect the existence of specific HPV genotype. In the heterogeneous solution, the operating staffs add the specimen DNA, genotype-specific RNA probe and the beads-coated with Ab into the reaction well in the correct order. Then eliminate the supernatant (including specimen DNA, non-captured RNA probe, beads and buffer) after the hybridization of RNA/DNA and the recognition of Ab is finished. Next is added the chemiluminescent substrate CDP Stare (Tropix PE, British) which is modified by the enzyme conjugated on Ab. After the EIA amplification reaction is complete, the chemiluminescence is detected by the luminometer.
According to research, DNA/RNA hybrid contains higher affinity than DNA/DNA hybrid and RNA/RNA hybrid. Besides this, another reason to choose DNA/RNA hybrid is base on its specificity. The DNA/RNA hybrid provides a higher accurate target for the Ab recognition than DNA/DNA hybrid in reality situation.
The HC2 is still a developing detection method. Some scientists are trying to make it better, especially in the HPV RNA-probe (12, 13). Other investigators are working to develop new techniques to the HC2. While a smaller number of researchers try to use old methods or new techniques in combination with HC2.
1-5. High-resolution melting analysis (HRM)
High-resolution melting analysis (HRM or HRMA) is not a totally brand-new technique. It’s an advanced application of real-time PCR. In fact, HRM and real-time PCR come from the same research group (University of Utah Medical School, Salt Lake City, USA), the leader of which is Prof. Carl. T. Wittwer (21, 34, 43, 45). Real-time PCR, also called quantitative PCR (Q-PCR) is actually a powerful and revolutionary technique which is denominated by the continuous and instantaneous quantification. It is able to detect the DNA concentration with the same species template in a single amplification reaction and show it in CT or CP value. SYBR Green is an important material in real-time PCR (18). It is able to bind the major groove of the DNA double-helix structure. And SYBR Green will emit a fluorescent light in the 483-583 nm range with the excitation of laser light of the real-time PCR instrument. In other words, the fluorescent light will represent the DNA concentration at each detective point of amplification process.
In the HRM amplification process, the saturated dye is an important and newly invented material which replaces SYBR Green dye in the amplification process (44). The saturated dye is not only able to bind DNA groove more tightly than non-saturated dye (SYBR Green) but also show higher sensitive in differentiating different length PCR products (44). As for Roche System, high-resolute dye (HRM dye, Roche, Germany) is the first choice compared with others, like LC Green I and LC Green plus. According to the development of high-affinity saturated dye and PC software, the HRM contains three analysis programs, melting plot, derivative plot and differential plot. Among these analysis plots, differential plot is a comparison value of fluorescent light between different reaction wells at the same time. Differential plot is a powerful tool to differentiate the point mutation, gene deletion, gene insertion and virus variants (24). The other two analysis plots, melting plot and derivative plot which are introduced in real-time PCR are the absolute value of fluorescent light in the analysis programs. However, HRM is a rapidly developing technique, with more and more scientific publications in the past few years. And a lot of applications will be discovered in the future.
1-6. Unlabeled oligoantisense (unlabeled probe)
For conventional real-time PCR, there are two type of fundamental fluorescent materials, dye (SYBR Green) and probe (TaqMan probe or hybridization probe). Both of these two materials were used for many years. In 2004, a new idea which combined the advantages of dye and probe was introduced (47, 48, 49). The characteristics of SYBR Green are low cost and extensive applications. The characteristics of TaqMan probe which could be designed for special sequence is expensive but contains the ability of target shooting. The design of unlabeled oligoantisense (unlabeled probe) is similar to that of the primer but is modified at the 3’ tail or 5’ head of oligoantisense by phosphorylation, C6-amino, inverted dT and the other chemical compounds. The unlabeled probe is added in the beginning of the amplification process and interferes with the extension step as finite depending on the lower annealing temperature comparison to the temperature of DNA extension. In the process, the unlabeled probe is similar to the TaqMan probe in that it can recognize the target sequence but without the emission of fluorescence. The fluorescence comes from the binding of saturated dye added in the beginning of the amplification process. In other words, the unlabeled probe is view as the second PCR product in the PCR process. In the past few years, the role of unlabeled probe had been proven in many scientific journals, like Herpes Simplex virus-1 and -2 (HSV-1 and HSV-2) differentiation, internal temperature control in amplicon melting genotyping and Apolipoprotein E (Apo E) genotyping (4, 32, 36). Till now, the application of unlabeled probe is still developing.
1-7. Asymmetric HRM
Asymmetric HRM is an application of conventional HRM. In comparison between symmetric HRM and asymmetric HRM, asymmetric HRM is denominated by its imbalance of products (38). In the beginning of amplification process, the excess primer is added 10- or 5- fold than the limited primer, and after the complete of amplification, there is one single-strand PCR product more than the other strand. Also, asymmetric HRM is a cooperative technique which combined with the unlabeled probe. The imbalance in PCR product is necessary because the spare single strand provides an annealing template for the unlabeled probe. After completion of the amplification process, an additional denaturing and annealing cycle is necessary for the probe to hybridize the template to create the second PCR product, and it provides the second detectable signal under the excitation of laser. Generally, there are two type of double strands produced, one is PCR product, and the other is the unlabeled probe induced hybridization product after the program is finished. With the binding of saturated dye and excitation of laser, there are two obvious viewpoints which represent the signals of PCR product and probe induced hybrid at two temperature grades in the analysis modes of melting plot and derivative plot (7).
Chapter 2: Methods
2-1. Clinical sample preparation
All of the clinical samples were collected from the patients of the department of obstetrics and gynecology in Chang-Gung Memorial Hospital (CGMH, Taiwan). All of the clinical swabs or surgery tissues were treat by medical technologists of the department of clinical virology. QIAamp DNA mini kits (Qiagen, Germany) were used to extract DNA of cervical specimens; the eluted DNA volume was 50 μl, and 2 μl of the aliquot was used for PCR amplification. Those female cervical specimens were prepared for HPV genotyping with the clinical method, EasyChip HPV genotyping system (KingCar, Taiwan). We also collected the surplus specimen to test the HPV detecting system, High resolution melting analysis (HRM).
2-2. Specimen DNA quality check and cloning preparation
The specimen DNA was amplified by conventional PCR, including quality control with GAPDH-specific primer and broad-spectrum primer for HPV L1 region. The protocol of GAPDH quality and HPV L1 amplification were complemented with 5 μl Mc buffer (10X), 2.0 mM MgCl2, 0.2 uM of each primer, 0.4 mM of each dNTP, 0.5μl Mc Taq, 2 μl template DNA and replenished to 50 μl with ddH2O. The amplification was optimized in the instrument (Veriti Thermal Cycler, ABI, USA)
The protocol of HPV L1 region amplification was carried out at 95 ℃ for 5 minutes, followed by 50 amplification cycles of 95 ℃ for 30 sec, 46℃ for 40 sec, and 72 ℃ for 30 sec. Next was extension for 3 minute at 72 ℃ for the replenishment of PCR product and cooling at 4 ℃. The step of GAPDH quality test was carried out at 95 ℃ for 5 minutes, followed by 50 amplification cycler of 95 ℃ for 30 sec, 60 ℃ for 40 sec, and 72 ℃ for 60 sec. Extension lasted 3 minute at 72 ℃ for the replenishment of PCR product and cooling at 4 ℃. After the amplification complete, the PCR product was analyzed with 2％ agarose gel.
2-3. High-resolution melting analysis: asymmetric and symmetric PCR
The HPV genotyping was performed by Roche Light Cycler 480 High-Resolution melting Master (commercial kit, Roche, Germany). There are two kinds of PCR included in the detective system of HPV genotyping, symmetric and asymmetric PCR. The protocol of symmetric PCR was complemented with 10 μl Roche HRM master (FastStart taq, HRM dye ,dATP, dGTP, dCTP and dUTP), 2.5 mM MgCl2, 0.5 μM of each primer, and 2 μl template DNA and replenished to 20 μl with ddH2O. The protocol of asymmetric PCR was complemented with 10 μl Roche HRM master, 2.5 mM MgCl2, 0.05 μM forward primer, 0.5 μM reverse primer, 0.5 μM oligoantisense, and 2 μl template DNA and replenished to 20 μl with ddH2O. The amplification was optimized for the use of HotStart Taq Polymerase in the instrument, a LightCycler 480 (LC480, Roche, Germany). The activation of enzyme was carried out at 95 ℃ for 15 minutes, followed by 65 (Symmertric PCR) or 75 (Asymmetric PCR) amplification cycles of 95 ℃ for 15 sec, 46 ℃ for 20 sec, and 72 ℃ for 30 sec, with the ramp to 95 ℃ at 4.8 ℃/s, to 46 ℃ at 2.5 ℃/s, and to 72 ℃ at 4.8 ℃/s. Extension of 1 minute at 72 ℃ was performed for the replenishment of PCR product, followed by increase to 95 ℃ at 4.8 ℃/s for the denaturing of PCR product and cool down to 55 ℃ at 2.5 ℃/s for hybridization. Finally, the melting plot quantification from 60 ℃ to 95 ℃ with 25 reading points for each degree of temperature.
2-4. Electrophoresis
To further confirm the results, those of including conventional PCR and HRM, we use 2％ agarose gel (2 g Agarose in 100 ml 1X TAE) buffer to detect the target PCR product length. The target HPV L1 PCR product was about 210 bp, and the GAPDH PCR products were 400 bp or 700 bp depend on which primer pairs.
2-5. Plasmid construction with TA-cloning
To further construct the HPV standard curve in HRM analysis, we had to have experimental materials which were equal to the reality HPV DNA and not used up in the construction process. We attempted to insert the target HPV region into the plasmid so that it would be easy to preserve and provide a stable experimental target. The HPV plasmids were cloned with different HPV genotype products which were amplified by the primer pair FRG-5-FRG-2 from the HPV L1 consensus region. The plasmid construction procedures were optimized by the commercial TA-cloning kit (RBC Bioscience Cloning System). After the plasmid construction was complete, we had 8 specific HPV plasmids, they are HPV16, 18, 31(42), 33, 39, 45, 52 and 58. Those plasmids were transformed into the HIT competent cell (DH5α, RBC Bioscience Cloning System). We select the white colonies which represented the success in both plasmid construction and transformation from the ampicillin-containing LB plate with the addition of 50 μl (50 mg/ml) X-gal.
The ampicillin-containing LB plate was prepared with 40 g LB agar powder in 1 liter ddH2O and sterilized in an autoclave. And the ampicillin-containing LB broth was prepared with 25 g in 1 liter ddH2O and sterilized in an autoclave. After the complete of sterilization, we added 1 ml ampicillin solution ( 100 μg/ml in 30％ EtOH ) into 1 Liter LB agar and broth (100 ng/ml). And the LB agar and broth were preserved in 4 ℃ refrigerator.
2-6. Plasmid DNA extraction and preservation
After the complete construction of HPV plasmids, we had to check the accuracy by DNA sequence. The extraction procedure was optimized with a commercial kit, High-Speed Plasmid mini kit (Geneaid). After the confirmation of HPV plasmids, we treated the HPV plasmids in two ways. Firstly, we preserved the HPV plasmid-containing cell (DH5α) with the addition of glycerol in a – 80 ℃ refrigerator. And we diluted the HPV plasmids with 10 mM Tris-HCl (pH 7.0) to 1 ng/μl as the preservation in a -20 ℃ refrigerator so that we could take it as needed.
The plasmid concentration was determined by an instrument, Nanodrop determinator (NanoVue Spectrophotometer)
Chapter 3: Results
3-1. Basic condition monitoring by conventional PCR
In the beginning, we got 8 clinical specimens from Chang-Gung Memorial Hospital (CGMH, Taiwan). Each tube represented one HPV genotype which was determined by commercial kit, EasyChip (KingCar, Taiwan) by the research group of Dr. Chyong-Huey Lai (CGMH). These 8 HPV genotypes were HPV16, 18, 31 (42), 33, 39, 45, 52 and 58. The first step in early stage when we received the specimen was quality check by conventional PCR with the primer pair of SPF1-GP6+ quoted from Dr. Lai’s laboratory of obstetrics and gynecology department (15, 16, 17). However, this operating procedure is just a quality test but did not match our experimental requests. Also, according to the gel electrophoresis result, those PCR products amplified by the primer pair of SPF1-GP6+ were impure and complicated (Fig. 1). The gel electrophoresis showed multiple, unregulated and unexpected PCR products which would interfere with the ability of our designed HPV genotyping system. But that is a predictable result because the forward primer consisted of 4 forward primers which contain a lot of degenerated nucleotides and Inosine. The primer pair which contains degenerated nucleotides and Inosine will have the lower recognition specificity in primer annealing. Based on this situation, the first thing when we attempted to design a new primer pair was to be as much as possible to eliminate the degenerated nucleotides and Inosine quoted from the published primer of the other research groups. We try to develop a new primer pair which is resembled but was a little different from those published primers.
The first primer pair FRG-1-GP6+ seemed to be a wonderful choice. But this primer pair contains a severe defect. It only amplifies a part of high-risk HPV (HR-HPV) genotypes, including HPV16, 18, 31 (42), 33, 45 and 52 (Figs. 2A, B). According to the experimental design, we tried to differentiate all HR-HPV in single reaction. Obviously, this primer pair is unable to distinguish all HR-HPV genotype in single procedure because the lost (missing) of a part of HPV genotype. In the following test, we have designed a few primers, including FRG-2,-3, -4, -5, -6, -7 and -8. The odd number means forward primer, the even number means reverse primer, and the larger number indicate it was designed later. The FRG-3 and FRG-7 were excluded in designed stage; the others were tested in reality. FRG-5, it was the first challenge in which we used the Inosine and a few degenerated nucleotides in our designed primers at the same time. It differs from the published primer pair SPF1; we attempted to add degenerated nucleotides and Inosine and eliminate the multiple PCR products. We hoped to have the advantages and excluded the disadvantages. As for these primers, the gel electrophoresis results showed me that the primer pair FRG-1-GP6+ could amplify HPV16, 18, 31, 33, 39, 45 and 52 (Figs. 2A, B). But the PCR products of HPV39 and 52 were weak, and HPV58 is un-amplified. It appeared that the primer pair FRG-1-GP6+ was unable to match the experimental aim. Actually, we had further designed another two reverse primers, FRG-2 and FRG-4, base on GP6+ at the same time. These two reverse primers contain two common degenerated nucleotides and one different degenerated nucleotide, the position of FRG-4 is near the 3’ tail, the position of FRG-2 is near the 5’ head. We hoped that these two primers, FRG-2 and FRG-4, can improve the amplification efficiency. Before we performed the experiments, we predicted that FRG-4 would be better than FRG-2 because of the primer tendency. However, in the following test, we made up three primer pairs, FRG-1-GP6+, FRG-1-FRG-2 and FRG-1-FRG-4. At this stage, we tested HPV39, 45, 52 and 58 with these three primer pairs and two magnesium concentration 2.0 mM and 3.0 mM at the same time. We had hoped to have a broad condition test so that we could get a better amplification condition as soon as possible. However, we couldn’t get an ideal result because the amplification efficiency was without improvement when the condition changed. In fact, it was exciting to get the PCR products of HPV52 and 58 with primer FRG-1-GP6+ only at magnesium 2.0 mM. But it was based on the triple-specimen DNA added, and the heavy primer dimer represented the low specificity and amplification efficiency (Figs. 3A, B, C).
However, we had two notions from these three primer pair tests; primer dimer elimination and the specificity rise of the HPV target region. Actually, these two notions could be reached with one step, forward primer re-design. We further analyzed the specificity and annealing affinity of each reverse primer, and we believed that the reverse primers including FRG-2 and FRG-4 were good enough to adapt 13 HR-HPV. We though the forward primer, especially the 3’ tail will be the problem. And we started considering extending the primer length because we couldn’t find another position better than the target site of FRG-1. Totally we had three steps to accommodate this primer, including the extension of primer length, the rise in number of degenerated nucleotides and the new addition of Inosine. We concluded the advantages of degenerated nucleotides and Inosine. We found one special position in which Inosine would be a better choice than degenerated nucleotide. The ideal primer, FRG-5, which contains one Inosine and three degenerated nucleotides is designed to challenge the experimental aim. At this stage, we had two primer pairs, FRG-5-FRG-2 and FRG-5-FRG-4. In the gel electrophoresis, FRG-5 showed higher annealing ability (48 ℃) than FRG-1 (46 ℃) in the combined tests with FRG-2 and FRG-4 (Fig. 4A). These two primer pairs could effective amplify HPV39, 45, 52 and 58 better than we did. Besides HPV45, the other three genotypes were unstable or low amplification efficiency in the previous tests (Fig. 4B). It was a breakthrough that we could amplify all 8 target HPV genotypes with one primer pair and single amplification reaction although it contained a few questions.
But we only needed one functional primer pair in HPV plasmid construction and HPV genotyping system. After we compared the amplification affinity of primer pairs, FRG-5-FRG-2 and FRG-5-FRG-4, FRG-5-FRG-2 seemed to be the better choice. It expressed higher PCR ability no matter whether it showed higher PCR concentration products of HPV39, 45 and 52 or the obvious HPV58 PCR product which was un-amplified with primer pair, FRG-5-FRG-4.
Besides the test of new primers, we also attempted to monitor the PCR condition. In particular the concentration of Mg2+ was a key factor to interfere with the efficiency of taq polymerase (5). After we confirmed the datasheet of the Mc Taq (Onestar, Taiwan), we tested the concentration of Mg2+ with 3.0 mM, 2.5 mM and the reference value 2.0 mM at the same time. Those chosen targets were HPV39 and HPV45 because these two HPV genotypes are not easy amplified. Firstly, we tested the HPV39 and 45 with the primer pair of FRG-1-GP6+. The result showed me similar results on 2.0 and 2.5 and 3.0 mM for HPV45, but lower amplification efficiency with the rise of Mg2+ concentration for HPV39 (Fig. 5A). After we considered the operating procedure and PCR result, especially HPV39, we defined the concentration on 2.0 mM. This defined concentration was confirmed with the primer pairs, FRG-5-FRG-2 and FRG-5-FRG-4 later (Fig. 5B). However, after we compared the previous results, 2.0 mM Mg2+ seemed to be an ideal choice despite the primer pair being changed.
3-2. Plasmid construction
The 8 HPV genotype PCR products which were amplified with the primer pair of FRG-5-FRG-2 were cloned into the plasmid for the stock that we can use it for the quality test (positive control), condition test and standard curve construction. In the operating schedule, those 8 genotype plasmids were completely constructed two different times because the HPV45 and 58 were contaminated the first time. However, we had constructed the plasmids of HPV16, 18, 31 (42), 33, 39 and 52 in the beginning. Secondly, we constructed the plasmids of HPV45 and 58. The first thing we did after the plasmid construction complete was amplified with primer pair FRG-5-FRG-2; it was an easy test to check whether the plasmid construction had succeeded or not. After the analysis of gel electrophoresis, we knew all 8 HPV plasmids had been successfully constructed (Fig. 6A). But, when we checked those genotypes, accuracy of HPV plasmid with the sequence information, I found a mistake. The sequencing information showed me that the plasmid of HPV31 (42) was incorrect (a few months later). After the recheck of sequencing and alignment with NCBI Genebank, the real genotype of the HPV31 (42) plasmid was HPV42 (Sup. 1). It was a big problem because the genotype information of the clinical specimen came from the Dr. Lai’s lab. It reminded me that I had to check the information of the specimens even if they had been determined by other methods. Besides the mistake with of HPV31 (42), we still had 7 HPV plasmids which represented the correct HPV genotype for the HPV stock. However, this mistake with HPV31 (42) is just an unexpected case that wouldn’t interfere with the others, and the standard operating procedure of standard curve construction was on schedule.
After we confirmed the really HPV genotypes of each plasmid, we performed a gradient PCR test with the constructed plasmid DNA by conventional PCR to check the amplification limitation. We chose HPV16, 18, 33 and 45 as the PCR gradient targets after the primary HRM test (data not shown. The primary HRM result expresses obvious difference in these 4 genotypes). In the gel electrophoresis (Figs. 6B-E), there were 210 bp PCR products around the concentration from 10-3 ng/μl to 10-6 ng/μl. We got a similar result with the limitation test in HRM test (Fig. 10A). According to the mathematical calculation, the concentration of 10-6 ng/μl represented 300 copies of plasmid. However, we had never tested the limitation, like 30 copies per micro-liter under 10-6 ng/μl at this stage because we thought it was enough. Actually, we didn’t know the amplification limitation in our HPV genotyping system at the moment. However, the limitation of conventional PCR was not the experimental aim, the differential curves of the high-resolution melting analysis were.
3-3. Standard curve construction with high-resolution melting
By 2009.5 (two thousand nine, May), we had successful constructed 8 genotypes HPV plasmid, they were HPV16, 18, 31 (42), 33, 39, 45, 52 and 58. In the following test, we attempted to construct the standard curve which could be contrasted with those of clinical samples in routine high-resolution melting analysis.
Firstly, we had to find the efficient DNA template concentration which resembled the real situation of HPV DNA in specimen sample and showed effective distinguishable curve. In early stage test, we had only successfully test two DNA template concentrations, 10-2 and 10-4 ng/μl (Figs. 7A, B). Those reactions of two template concentrations had similar Tm value, specific curve and regular CT value despite the concentration of 10-4 ng/μl expressing less clearly than the concentration of 10-2 ng/μl. After a series of DNA concentration gradient tests (Figs. 6, 7), we set up the ideal concentrations of each HPV plasmid DNA between 100 pg/μl and 10 pg/μl. After we got the efficient DNA template concentration, we attempted to construct the standard curve of each HPV genotype with this ideal concentration in the following test.
After a series of tests, we had constructed the standard curve in derivative plot of HRM. We wanted to emphasize the derivative plot because we selectively excluded the third mode, the differential plot in HRM (See Discussion). According to the derivative plot, each genotype plasmid had a specific curve that we could differentiate them from each other. Otherwise, 8 curves of derivative plot represented 8 HPV genotypes. These 8 HPV genotypes were classified into two groups, the single-peak group (HPV18, 39, 45) and non single-peak group (HPV16, 31 (42), 33, 52 and 58) (Figs. 8A, B). However, after we considered the real situation of clinical samples, the characteristics of 8 genotype plasmids were not always strong enough.
It was easy to distinguish the staircase or non single-peak group in this stage, but the single-peak group was too close to determine from each other. The only one distinguishable point in the single-peak group is the difference of Tm value. In fact, it would be a poor situation in clinical sample determination if we only used the normal HRM. So we introduced a new material to increase the differential ability, the oligoantisense (unlabeled probe) into the amplification process. We hoped it would provide another functional signal with the addition of unlabeled probe to improve HPV differentiation. With this additional material, the amplification condition should switch the symmetric PCR to asymmetric PCR in HRM HPV genotyping system.
We chose one HPV plasmid as the confirming test to check the difference since we had decided to apply asymmetric HRM. Fortunately, the standard curves between two types HRM are alike (Fig. 9) (Data show HPV16 only). We even got an exciting result that the minor curves of HPV16, 33, 52 and 58 were clearer in asymmetric HRM than in symmetric HRM. Otherwise, the single-peak group expressly showed the identical characteristic as same as symmetric HRM.
Since getting the conclusion that there is no expressly difference between symmetric and asymmetric HRM, we have performed a series experiments, including the template limitation test, multiple unlabeled probes application and the clinical specimen test. We chose HPV18 as the experimental target from the single-peak group to perform the gradient asymmetric HRM test with HPV18-specific unlabeled probe. Based on the lower amplification efficiency of asymmetric HRM, we extended the PCR cycle from 50 to 65 cycles so that we could as best as possible to amplify those of samples with low-concentration templates (Fig. 10A). The result showed a constant delta CT value in amplification plot and the identical characteristics in derivative plot and melting plot (Figs. 10B, C). With decrease of template concentration, there was no obvious difference in derivative plot after the amplification procedure was completed. Also, the specific curve induced by HPV18-specific unlabeled probe between 70 to 75 ℃showed the effective signal which we could differentiate from those of HPV39 and 45. After the primary confirming test with HPV18, we had constructed the standard curves like we did before.
The characteristics between symmetric and asymmetric HRM were alike. Therefore, there is no obviously difference except the unlabeled probe-induced curves. In the standard curve re-construction in asymmetric HRM, we used two genotype-specific unlabeled probes in single reaction, the HPV16- and HPV18- specific probes. The reason that we set up these two probes was that have the highest epidemiologic percentage in the world. In most countries, HPV16 and 18 totally occupy over 70％ of clinical cases (28). After the addition of HPV16- and 18- specific unlabeled probes in the same amplification, the other 5 HPV genotypes plasmids {exclude HPV31 (42)} expressed no difference. HPV16 induced a signal between 63 to 68 ℃ and HPV18 shows the signal between 70 to 75 ℃ (Figs. 11A, B). However, the standard curves of asymmetric HRM were clearer than those of symmetric HRM, so we can more easily to differentiate these 7 HPV genotypes.
The same as we did before, we also had a few tests including the HRM condition test, template mixing test and some interesting experiments. For example, regarding the concentration of Mg2+, we consulted the reference value of the conventional PCR. However, 2.0 mM Mg2+ is just a property value in conventional PCR but not always correct in HRM. We tested the concentrations of 2.0 and 2.5 mM at the same time. We found that 2.5 mM was the better choice in HRM because of the higher amplification efficiency (Figs. 12A, B). It was an obvious result that the CT value was lower at 2.5 mM than 2.0 mM. Furthermore, there is a special phenomenon that a constant Tm value shift exists. Not only HPV16, but also other HPV genotypes contain this characteristic (Data not shown). Therefore, 2.5 mM Mg2+ in HRM will be a wonderful choice in the following experiments
After the complete construction of standard curves within HPV genotype in asymmetric HRM, we collected more than 1000 clinical specimen samples from the Department of Clinical Pathology to detect the HPV genotype in asymmetric HRM.
3-4. Clinical test
At first, we used the conventional PCR with GAPDH primer to amplify the specimen DNA to check the sample quality (data not shown). This was a necessary step in order to make sure the quality of clinical specimens was satisfactory. Totally, we had tested about 140 clinical specimens in this procedure. After the quality check, we identified the genotype of 140 specimens in asymmetric HRM within 4 rounds.
In clinical samples undergoing HRM test, we separated all 140 specimens into a few groups, including target genotypes (HR-HPV), non-target genotypes (non-HR-HPV), non-amplified cases, HR-HPV variants and HR-HPV candidates. Among these clinical samples, we found 6 standard genotypes, HPV16, 18, 39, 45, 52 and 58 but not HPV33. We also found 3 new HR-HPV candidates which were confirmed by DNA sequencing as belong to HR-HPV; they are HPV51, 56 and 68. Also, the plasmid construction procedure was on schedule. Besides these 10 defined HR-HPV genotypes, there were a lot of unknown HPV genotypes found in HRM tests. We temporary denominated them as genotypes A, D, F, G and H.
In fact, we sent a few cases to be DNA sequencing. Unfortunately, we didn’t distinguish genotypes A, D, F and H very well because of the close contact of the curve according to derivative plot in the beginning. All of these cases didn’t belong to HR-HPV and were inconsistent. As for those inconsistent results, we classified them as the non-target genotypes. We confirmed the DNA information again and again, and we further checked the clinical data a few months later (Sup. 5). Finally, we had confirmed the really genotypes of these non-target genotypes. Actually, most of these non-target genotypes are LR-HPV and subtypes. Among these non-target genotypes, we found a few conserved clinical cases, like CP8304 (genotype A), MM8 (genotype D), CP8061 (genotype F), HPV62 (genotype H) and a few unique cases, like HPV54, 70, 71, 80, 81 and LiAE5. However, there are no HPV35 and 59 in these 140s clinical samples, and we can’t determine HPV31, 33 and 51 very well. Therefore, the performance of HPV35 and 59 determinations and the improvement of HPV31, 33 and 51 amplifications will be the next topic.
Chapter 4: Discussion
After a long term experiments, we have successful determined 10 HR-HPV genotypes in our HPV genotyping system, including HPV16, 18, 33, 39, 45, 51, 52, 56, 58 and 68. There have been a lot of devises to detect these 10 HR-HPV genotypes in the past two years. From the primer design and PCR condition rearrangement to HRM condition monitoring and the application of unlabeled probes, each series of tests represents different significances. However, there are a few questions that we still can’t solve well. Also, we have prepared a few ideas on the advanced development of our HPV genotyping system. Here, we collate all of the conclusions from results, solved subjects, unsolved subjects and disposed designs that we will perform in the future experiments.
4-1. Specific genotype analysis
There are a lot of interesting stories in the construction of our HPV genotyping system; each story represents a mistake or a new discover in the process of setting up the system. In these HPV genotypes, HPV31 (42), 33, 39, 45, 51 and 68 are the most important to discuss, especially HPV51, 56 and 68. Despite the standard operating procedures were not completely finished. We have discussed them in the following subtopic.
4-1-1. HPV31 and 42
HPV31 (42) was an operating mistake because we had missed the real genotype of HPV31 (42) plasmid. HPV31 (42) is one of the 8 original HR-HPV genotypes that we got from an assistant in Dr. Lai’s lab. However, we never queried the information until the construction of the 8 HPV genotype plasmids was completed. At first, we confirmed the DNA information of the 8 HPV genotype plasmids with the Vector NTI alignment program. Each plasmid represents a unique genotype, and we didn’t check the real genotype at that time, waiting instead until a few months later, when the standard operating procedure was almost complete. We checked the accurately genotypes of plasmid with the derivative plot of HRM, Vector NTI alignment program and NCBI gene bank. We further found one strange phenomenon; that HPV31 (42) didn’t match the HPV31 DNA sequence in the NCBI gene bank. The information of the NCBI gene bank indicated the highest matched HPV genotypes of HPV31 (42) plasmid was HPV42 (81％). And we have sent the HPV31 (42) plasmid for DNA sequencing in twice and confirmed the DNA sequence with alignment program and NCBI gene bank again and again (Sup. 2). We got an unexpected conclusion, that HPV31 (42) plasmid may not belong any one specific HPV genotype of those anticipative genotypes, no matter HPV 31, 42 or the other HPV genotypes. However, the closest genotype was HPV42 so that we temporary determined the plasmid as HPV31 (42) at the moment. After a long-term confirming test, we had compared the curves of derivative plot of HPV31 (42) plasmid and HPV42 specimen. They were perfectly matched each other, so we know HPV31 (42) was HPV42.
As for HPV31, we analyzed the primer information and HPV31 clinical data which were determined by EasyChip. There were HPV31 in our 140s clinical cases. We further found the primer pair, FRG-5-FRG-2, may be unable to amplify HPV31. Therefore, we re-designed a new primer pair and a HPV31-specific forward primer. We attempted to set up a new primer pair to represent the present primer pair, or a newly primer pair system, one broad primer pair combined with a genotype-specific primer in our HPV genotyping system.
4-1-2. HPV33
HPV33 was a strange phenomenon because we never found it in clinical sample tests. After we checked the existence of HPV33 in 140s clinical samples, we actually got a few HPV33 cases. But we couldn’t amplify it from the beginning to the end. Therefore, we further checked the primer pair; we found the reverse primer was weak to recognize the target region of HPV33 (Sup. 1). It means this primer pair was unable to amplify HPV33 very well. Otherwise, the correctness of HPV33 plasmid was plus so that it was a problem in reverse primer specificity. There is a weakness in present primer pair. Therefore, we treated this problem like we did in HPV31 (42).
We re-design a HPV33-specific reverse primer and a brand new broad-spectrum primer pair which combine those problems we face so far, including HPV31, 33 and the following problems we haven’t mentioned yet, like HPV35, 51 and 59.
4-1-3. HPV39
The story of HPV39 is multiple HPV39 variants. After comparison of HRM result and EasyChip result, we found some concordant and some discordant results (Sup. 5). In clinical tests, we only found a few cases which completely matched the curve of the HPV39 plasmid (HPV39 plasmid form, old form, HPV39A) in derivative plot. Additionally, we also found other conserved cases which were the major cases and different to HPV39A. Those new discovered conserved genotypes were determined as HPV39 B form (new form, HPV39B) (Sup. 3).
According to the differentiation of the derivative plot, HPV39B belong to the single-peak group. However, it was thought to be the other HR-HPV in the beginning because of the unique Tm value. The Tm value of HPV39B is located at the left side of HPV39A. After we send a few cases of this newly discovered HPV genotype (HPV39B) for DNA sequencing, it was determined as HPV39, and it showed high similarity with the HPV39A. In comparison of the partial sequence between HPV39A and HPV39B, we discovered 5 SNP in this 210 bp region. When we analyzed the 5 SNP, HPV39A contained 4-GC and 1-AT, and HPV39B contained 1-GC and 4-AT. From the ratio of GC and AT, we predicted that the Tm value of HPV39A higher than that of HPV39B. In fact, the clinical results confirmed our prediction (Figs. 7, 11). Therefore, HPV39 was the first discovered HPV variant our HPV differential system.
As for this situation, there are two possible ways to solve this problem. Firstly, we can construct the HPV39B plasmid as the second HPV39 plasmid standard because we only found two HPV39 variants, and each HPV39 variant was conserved and detectable. Second, we could design the HPV39-specific unlabeled probe. It’s a functional way to solve the HPV39 variant because we had done it on HPV16 and 18. However, we preferred the first way because we had only found two HPV39 variants so far. If we added the HPV39-specific probe in the amplification process, we had to consider the interference of HPV16 and 18 probes. Furthermore, we must collect more HPV39 clinical data to confirm the experiments because the few cases of HPV39A. The data of EasyChip may be wrong and just a coincident result to match HPV39A. In other words, HPV39A and the few cases of HPV39 in 140s clinical sample may both wrong. And the most important thing is that we have found the other HPV genotype target which is more worthwhile to apply this way. Whatever, we believe the HRM differential system is strong enough to detect two HPV39 variants even it may be unnecessary.
4-1-4. HPV45
HPV45 was thought to be the biggest problem in clinical specimen test. It’s not like the mistake of HPV31 (42) or the pure and conserved SNP of HPV39. According to the DNA information (Sup. 4), we have found more than 10 HPV variants around 5 to 6 point mutations. In clinical specimen test, we had detected a few unknown cases which were determined as HPV45 by DNA sequence. Besides this, we also found some un-determined cases which were close to HPV45 standard curve and determined by EasyChip. It means that we could not easily distinguish these HPV45 variants (HPV45v) by our HPV genotyping system. However, because of running out of HRM kits, we could not perform the second confirming tests. But we can predict that we can’t solve the HPV45 variants the same as HPV39 variants, because HPV45 variants are too much to differentiate in a single reaction. The material, the HPV 45-specific unlabeled probe will be a good choice.
In our HPV genotyping system, we attempted to decrease the amount of unlabeled probes as possible as we could; despite it seems that HPV genotype-specific unlabeled probes would not interfere with each other so far. However, we are the first research group to apply multiple unlabeled probes in single reaction of HRM. Those published papers have not yet indicated what will happen if we add too much unlabeled probe in a single reaction. So we retain the possibility that it will induce some unpredictable problems. However, HPV45v will be a better target than HPV39v for applying this technique.
4-1-5. HPV51
HPV51 was discovered in clinical specimen test, too. It belongs to the single-peak group. Actually, the Tm value of HPV51 is located at the worst position (80℃), in that a lot of unknown and un-specific HPV genotypes are there. We sent a lot of clinical cases which are locate at about 80℃ for DNA sequencing. We found a few HPV genotypes around there, including HPV51, 70, 71 and 81. Most of these cases did not belong to HR-HPV. But we have not yet checked the derivative curve of HPV51 again because of running out of HRM kit, and we only found one HPV51 case so far. In fact, the epidemiologic evidence indicates that HPV51 is less than 1％, but there are more than 5 HPV51 cases in 140s specimens (6). It was an abnormal phenomenon to find so many HPV51. Therefore, we must consider: what if the clinical data of EasyChip is incorrect. However, we must collect more data so that I can make a reasonable and correct decision.
4-1-6. HPV56
HPV56 was discovered in clinical samples, too. Despite it belongs to single-peak group, it was more easily differentiated than the other single-peak HPV genotypes because of its isolated position in the derivative plot. The Tm value of HPV56 is 77℃; it’s locates at the left end in these 10 well-known HR-HPV genotypes (data not shown). Till now, we have found more than one clinical sample, and the plasmid construction was complete and ready to work.
Based on the specificity of HPV56, we don’t have to do any special treatments like HPV39 variants and HPV45 variants.. The special and isolated Tm value of HPV56 is a unique characteristic, so that we can differentiate it from the other well-known genotypes.
4-1-7. HPV68
HPV68 is one of the three newly discovered HR-HPV genotypes, but it belongs to double-peak group. In fact, we thought HPV68 was the mixed genotypes of HPV33 and 18 in the beginning, because HPV68 consists of the minor curve of HPV18-specific unlabeled probe-induced curve and the major curve of HPV33. But after comparison of the minor curve of HPV18 unlabeled probe-induced curve and the minor curve of HPV68. We discovered that the temperature range of HPV18 unlabeled probe is bigger than the other well-known minor curve of the other double-peak HPV genotypes. So we predicted that it may be a new HPV genotype. Fortunately, after the confirming of DNA sequence information, it was a newly discovered HR-HPV, HPV68. Like the other double-peak HPV genotypes, HPV68 contained the strongly distinguished curve which would be an obvious characteristic different to the other HR-HPV.
4-2. Summary
In clinical specimens, we found a lot of unknown and conserved HPV genotypes. Among these cases, genotypes A, D, F and H were thought to be the candidates of HR-HPV according to the highly conserved derivative curves and high amount of cases in the beginning. However, after the confirming of EasyChip data and DNA sequence information, we know the real genotypes of A (CP8304), D (MM8), F (CP8061) and H (HPV62). All of they are not HR-HPV. Besides this, there are a lot of cases which we can’t amplify or determine them well. Therefore, the EasyChip data in 140s clinical samples is a good reference to help us to eliminate some unimportant cases and remind us those important and missing cases in round one test. And we will re-check those missing cases, like HPV31, 33 in the following tests.
Till now, we have differentiated 10 HR-HPV genotypes in single reaction. In epidemiologic statistic of Taiwan, these 10 HR-HPV represent over 90 ％ cervical cancer cases in Taiwan (19, 20). If we analyze this HPV genotyping system (asymmetric HRM with unlabeled probes) by ourselves, we think the sensitivity is good enough. In opposite, the specificity is lower because there are too much HPV genotypes exist in clinical sample, including HR-HPV and LR-HPV. So, the first mission of ours is collecting more clinical data as many as possible that we can tell people this HPV genotyping system is functional and powerful.
If we can successful determine 13 HR-HPV, the multiple HPV genotypes infection will be the next topic. This topic is another big challenge because PCR-based method is usually unable to differentiate multiple-types DNA in single reaction with the same amplification procedure and protocol. However, we still attempt to use the same technique to solve this topic.
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