Wudpecker Journal of Medical Sciences Vol. 2(6), pp. 053 - 057, December 2013 ISSN 2315-7240 2013 Wudpecker Journals Evaluation on the amplification of HPV and EGF genes by loop-mediated isothermal amplification Lishi Wang1,2,3, J. H. Maeng#, and E. K. Lee2#* 1 Department of Orthopedics Surgery, University of Tennessee Health Science Center, Memphis, TN 38163, USA. Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, Ansan, Korea 426-791. 3 Department of Basic Medicine, Inner Mongolia Medical University, Inner Mongolia, 010110, P.R.China # College of Bionanotechnology, Gachon University, Seongnam, Korea 461-701 2 # *Corresponding author E-mail: eklee@gachon.ac.kr. Tel: 82-31-750-8982, Fax: 82-31-750-8769. Accepted 25 September 2013 Loop-mediated isothermal amplification (LAMP) is a newly developed gene amplification technology. In this study, we applied the LAMP method to amplification of hEGF (epidermal growth factor) and HPV (human papilloma virus) genes to compare the result with that from the conventional PCR for efficiency. We also applied these two methods to a mixture of DNAs. The comparisons showed that LAMP was simpler and more efficient than the PCR. PCR amplification often has background noises or nonspecific amplification in some cases; while in this aspect, LAMP reaction is more sensitive and specific. Furthermore, due to its easy manipulation, LAMP technology could allow DNA amplification and gene detection in small clinics, clinical laboratories, small research laboratories and on-site forensic applications. Key words: Isothermal amplification, PCR, rhEGF, HPV66, DNA detection. INTRODUCTION Gene amplification is widely used in the upstream molecular biotechnology, and polymerase chain reaction (PCR) has been the most popular and widely used gene amplification method (Yang and Rothman, 2004; Espy et al., 2006).However, PCR has its own disadvantages; a laboratory has to be equipped with a thermal cycler and skillful manipulations which might limit its use. The whole round requires 2 to 3 hr because of temperature excursion for denaturation and annealing. To circumvent this, Notomi et al. (2000) have developed a new method named loop-mediated isothermal amplification (LAMP) that can provide 10 9-10 10 times DNA amplification in about 1 hr. This method, because of its simplicity and specificity, has brought great benefits to molecular biotechnology applications including clinical microbiological detections (Seki et al.,2005; Minami et al., 2006; Cho et al., 2006; Torigoe et al., 2007) and detection of infectious diseases (Bista et al., 2007; Toriniwa and Komiya, 2006; Tomlinson et al., 2012). In this study, we applied the LAMP method to amplification of hEGF (human epidermal growth factor) and HPV (human papilloma virus) genes. The result was compared with that of the conventional PCR method for efficiency. Furthermore, the LAMP method was applied to a mixture of DNAs to show its high specificity.EGF gene PCR product of 171bp was cloned into pGEM-T Vector (Promega, Madison, WI, USA) and then transformed into E.coli DH5α pTXB1 (New England Biolabs, USA). HPV66DNA samples (whole sequence was 7,824bp) were purified from human serum of an infected patient (Celltek Co., Ansan, Korea).HPV66 was reported to be probably the high-risk sexually transmitted HPV that may be the major cause of invasive cervical cancer (Tawheed et al., 1991; Clifford et al., 2005). Thus, highly sensitive and rapid detection of HPV66 is important to detect and prevent the disease transmission. For LAMP reaction, we designed two sets of primers for each of EGF and HPV66 genes (Table1).Amplification was carried out with a Loopamp DNA amplification kit (Eiken Chemical Co. Ltd., Japan). PCR was conducted TM with Minicycler 3000 (MJ Research Inc., USA).In the LAMP reaction, the use of 4 primers to recognize 6 distinct regions on the target gene sequence ensured high specificity of gene amplification. It was reported when 2 more primers, termed loop primers, were added, the LAMP reaction time could be shortened to 35 TO 40 min (Nagamine et al., 2002). In some cases, the primer design is very challenging. For this circumstance, stem Wang et al. primers were recently developed (Gandelman et al., 2011). RESULTS AND DISCUSSION Compared to PCR, LAMP method was superior in simplicity and efficiency in EGF gene amplification. The use of four primers to recognize six distinct regions on the target EGF sequence in the initial LAMP steps and two inner primers (FIP and BIP) in the subsequent steps ensures the high specificity of EGF gene amplification. In the case of conventional PCR reaction, an inhibitory effect might be present during the latter part of cycling, while LAMP reaction appeared to be limited only by the amount of deoxynucleotide triphosphates and primers. In the process of LAMP reaction, a large amount of pyrophosphate ions was produced, which reacted with magnesium ions in the reaction mixture to form magnesium pyrophosphate, a white precipitate byproduct. Due to its turbidity, the precipitate can be easily identified with naked eyes, as shown in Figure 1(a). Following the two amplifications of EGF gene by using LAMP and conventional PCR, we centrifuged the sample reaction tube and PCR reaction tube at 4,500rpm for 3 min. The LAMP products tube can be seen clearly with magnesium pyrophosphate as the by-product precipitate, while the reaction solution in the conventional PCR tube was clear with no precipitate. This turbid precipitate phenomenon allowed easy and rapid visual detection that the target DNA was amplified by LAMP, which composes one of the characteristics of LAMP. The turbidity would be intensified with the LAMP reaction cycling time and thus the amount of DNA synthesized. This feature enables the real-time monitoring of the LAMP reaction by real-time measurement of turbidity. In Figure 1(b), we successfully amplified EGF gene by LAMP method, as shown in Lane 2 and 3, aliquots of 5μL of EGF LAMP products were loaded in the loading wells of 1.3% agarose gel (stronger under UV light). The products appeared as a ladder pattern, with bands of different sizes from 130bp up to the loading well. Lane 1 is the negative control with no template DNA in the reaction solution. Lane 4 is the positive control DNA LAMP fragments. Detailed information about this positive control DNA is available from the Eiken Company (http:// loopamp.eiken.co.jp). To confirm EGF LAMP results, the amplified products were digested with restriction enzyme AluI. We chose this enzyme because in the restriction sites map of 171bp EGF gene sequence, there is only one AluI site located at position 76bp which can split the LAMP polymerized bands into two fragments of 90bp and 130bp. Thus, if the amplified EGF products had the exact structures, the product, after electrophoresed in 1.3% agarose gels 054 would present 90bp and 130bp fragments. Figure 1(c) shows the exact bands of 90bp and 130bp in Lane 3 according to the NEB DNA Marker. HPV66gene amplification: Human papillomavirus is a papillomavirus that infects the epidermis and mucous membranes of humans. HPV can lead to cancers of the vagina, vulva, anus, and cervix in women. Over 100 distinct types of HPV have been identified, and approximately 30 to 40 HPV types infect the anogenital region through sexual contact. It is reported that type 66 is probably the high-risk sexually transmitted HPV and may be the major cause of invasive cervical cancer. Therefore, highly sensitive and rapid detection of HPV is important to prevent transmission and monitor treatment. We amplified HPV66 gene by LAMP method. To confirm the LAMP results, the amplified products were digested with a restriction enzyme AluI. Figure 1(c) shows the electrophoretic result of the amplifiedHPV66 by LAMP and PCR. Lane 1 indicates the LAMP product of HPV66 gene. (Lane 2 is the LAMP product of EGF gene as a contrast). We could observe that the LAMP products for HPV66 gene appeared as a ladder pattern. Lane 3 and 4 indicate LAMP products of HPV66 gene after digestion with AluI. Restriction enzyme AluI can divide HPV66 LAMP product into three fragments of 224bp, 188, and 206bp. These results suggest that HPV66 gene was successfully amplified by LAMP method. Lane 6 is PCR product of HPV66 gene. (Lane 5 is PCR product of EGF gene as a contrast). To investigate the specificity of LAMP method, we applied LAMP and PCR method to DNA mixture. We fixed the LAMP reaction time at 1 hr, since the reaction was completed in about 1 hr regardless of the starting concentrations of DNA. LAMP and conventional PCR were performed with different mixture ratios of HPV66 and pDNA. As seen in Figure 2(a), HPV66 gene was successfully amplified by LAMP method in the presence of pDNA. In the case of conventional PCR, HPV66 gene was amplified well in the absence of pDNA. However, DNA amplification was interfered by the presence of unrelated pDNA (Figure 2b). Therefore, LAMP can be used for forensic applications, for example, when a gene in a minute amount of complex specimens needs to be amplified easily and rapidly. To compare the yield and purity of the LAMP method, LAMP and PCR was performed with the same concentrations (10ng/μL) of EGF and HPV66. The absorbance ratios of 260/230 (DNA/humic acids) and 260/280 (DNA/protein) were determined. As seen in Table 1, the yields of amplified EGF and HPV66 gene by the LAMP method were significantly higher than those by PCR. Moreover, the absorbance ratios of the LAMP product were between 1.8 and 2.0, indicating little or no protein contamination. LAMP, because of its remarkable characteristics of amplifying nucleic acid with high 055 Wudpecker .J. Med. Sci. Figure 1. EGF and HPV66gene amplification by LAMP and PCR. (a) Visual detection after centrifugation at 4,500 rpm for 3 min. APCR products tube with no pyrophosphate precipitate (left tube); a LAMP product tube with pyrophosphate precipitate (right tube); (b) Electrophoretic image of the LAMP-amplified product, Lane M, NEB 100bp DNA ladder; Lane 1, Negative control (with no template DNA); Lane 2 and3, EGF gene amplified by LAMP; Lane 4, LAMP product of positive control (template DNA and primer mix were supplied by Eiken Co. Ltd.); (c) Electrophoretic analysis of the HPV66 LAMP and PCR amplified products. Lane 1, HPV66 LAMP product; Lane 2, EGF LAMP product; Lane 3 and 4, complete LAMP products of EGF and HPV66 genes after digestion with AluI; Lane 5 and 6, PCR products of EGF and HPV66 genes. LAMP reaction:The reaction volume was 25μL by mixing 1.6μM each of FIP and BIP primers, 0.2μM each of F3 and B3 primers, 12.5μL of 2×Reaction Mix and a certain amount of template DNA (EGF or HPV66). The same reaction mixture without template DNA was used as a negative control. As a contrast, a positive control reaction was also used.The mixture was heated at 95℃ for 5 min, and cooled on ice, then 1μL of Bst DNA polymerase was added, followed by incubation at 63℃ for 1 hr and heating at 80℃ for 10 min to terminate the reaction. PCR reaction: The total volume was 25 μL with the outer primers F3and B3 as PCR primers, 400μM dNTP, 10×Taqbuffer 2.5μL, a certain amount of template DNA, and 1u Taq polymerase (New England Biolabs, USA). After the initial denaturation step at 95℃ for 5 min, thermal cycling at 95℃ for 30s, 62℃ for 30s and 72℃ for 30s was repeated 28 times.Electrophoresis: LAMP and PCR products were electrophoresed in 1.3% agarose gel followed by staining with ethidium bromide (EB). The gel images were acquired using Chemi-doc image analyzer (Bio-Rad, Hercules,CA, USA). Primer sequence sets used to amplify EGF and HPV66 genes: hEGF: F3 5’-ATGAACTCTGACTCCGAATGC-3’ (171bp) B35’-CAAGATCTTACGCAGTTCCC-3’ FIP5’-AACACCATCATGCAGGCAATCCGCTGTCTCACGACGGTT-3’ BIP5’-TACATCGGTGAGCGTTGCCAACCATTTCAGGTCGCGATAC-3’ HPV66: F35’- GGT CCA TGA TTA CCT CTG AG -3’ (216bp) B35’- ATT CCT CCA CAT GGC GAA -3’ FIP5’- ACAAATACCTGATTACCCCAGCATAAACCTTATTGGTTGCAACGT-3’ BIP5’- TGTTGTGGATACTACCAGAAGCACTTGATTTCACGGGCATCA-3’ Figure 2. The specificity test of: (a) LAMP and (b) conventional PCR for the HPV66 gene. Lane 1 and 5, HPV66 without another DNA; Lane 2 and 6, the mixture ratio of HPV66/pDNA of 1.5/0.5 (w/w); Lane 3 and 7, the mixture ratio of HPV66/pDNA of 1/1 (w/w); Lane 4 and 8, the mixture ratio of HPV66/pDNA of 0.5/1.5 (w/w). Wang et al. 056 Table 1. Yield and purity comparison between LAMP and conventional PCR products. Conc. (ng/μL) Abs 260/280 Abs 260/230 LAMP EGF 3,296 1.78 1.88 HPV66 2,912 1.81 1.91 specificity, efficiency and rapidity under isothermal conditions with a set of four (six) specially designed primers that recognize six (eight) distinct sequences of a target DNA, has brought great benefits to applied biosciences. ADDITIONAL COMMENTS Primer design and kit components performance Since the annealing of the four primers to the target DNA in the LAMP reaction for EGF and HPV66 was critical, we designed the melting temperature (Tm) of the four primers 0 to be within 58 to 62 C. GC contents were about 50-60% rich.It was reported that when two more primers, termed loop primers, were added, the LAMP reaction time could be shortened to 35 TO 40 min. In that procedure, six primers recognize eight distinct regions on the target DNA (Nagamine et al., 2002). In the LAMP reaction, Bst polymerase is another critical factor for efficient amplification because of its strand displacement activity. In the 2×Reaction Mix, the component betaine (1.6M) has the activity of reducing base stacking and destabilizing the target DNA helix and therefore was found to dramatically contribute to the amplification efficiencies. We used the negative control as a reaction contrast because LAMP reaction was very sensitive. On the other hand, if LAMP reaction was so sensitive that a template cross-contamination took place, the emergence rate of the false-positive products would be very high. Therefore, careful precautions against the template crosscontamination must be taken during sample collections and preparations for LAMP. Ever since the development of LAMP method, it has been used in basic research from a variety of perspectives. In addition, LAMP is also applied in a wide range of fields, including SNP typing and quantification of template DNA. This method is expected to be a more reliable and more widely used DNA amplification method. ACKNOWLEDGEMENT This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2008-0061855). PCR EGF 264 1.76 1.80 HPV66 471 1.79 1.79 REFERENCES Bista BR, Ishwad C, Wadowsky RM, Manna P, Randhawa PS, Gupta G, AdhikariM, Tyagi R, Gasper G, Vats A (2007). Development of a loop-mediated isothermalamplification assay for rapid detection of BK virus. J. Clin. Microbiol., 45(5): 1581-1587. Cho HS, Kang JI, Park NY (2006). Detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification. J. Vet. Diagn. Invest., 18(1):81-84. Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G, Pimenta JM(2005). Human Papillomavirus Genotype Distribution in Low-Grade Cervical Lesions: Comparison by Geographic Region and with Cervical Cancer. Cancer Epidemiol. Biomarkers Prev., 14(5): 1157-1164. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD,Wengenack NL, Rosenblatt JE, Cockerill FR 3rd, Smith TF(2006). Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin. Microbiol. Rev., 19(1):165-256. Gandelman O, Jackson R, Kiddle G, Tisi L(2011).Loopmediated amplification accelerated bystem primers. Int. J. Mol. Sci.,12(12):9108-9124. Minami M, Ohta M, Ohkura T, Ando T, Torii K, Hasegawa T, Goto H(2006). Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori. J. Clin. Microbiol., 44(11):4032-4037. Nagamine K, Hase T, Notomi T (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probes. 16(3): 223-229. Notomi T, Okayama H, MasubuchiH, Yonekawa T, Watanabe K, Amino N, Hase T(2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28(12): E63. Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M (2005). Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae. J. Clin. Microbiol. 43(4): 1581-1586. Tawheed AR, Beaudenon S, Favre M, Orth G(1991). Characterization of Human PapillomavirusType 66 from an Invasive Carcinoma of Uterine Cervix. J. Clin. Microbiol., 29(11): 2656-2660. Tomita N, Mori Y, Kanda H, Notomi T(2008).Loop- 057 Wudpecker .J. Med. Sci. mediated isothermal amplification(LAMP) of gene sequences and simple visual detection of products. Nature Protocols. 3:877-882. Tomlinson JA, Ostoja-Starzewska S, Adams IP, Miano DW, Abidrabo P, Kinyua Z, Alicai T, Dickinson MJ, Peters D, Boonham N, Smith J(2012).Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease. J. Virol. Methods.pii: S0166-0934(12)00256-X. doi: 10.1016/j.jviromet.2012.07.015. [Epub ahead of print]. Torigoe H, Seki M, Yamashita Y, Sugaya A, Maeno M (2007). Detection of Haemophilus influenzae by loop- mediated isothermal amplification (LAMP) of the outer membrane protein P6 gene. Jpn. J. Infect. Dis., 60(1): 55-58. Toriniwa H, Komiya T (2006). Rapid detection and quantification of Japaneseencephalitisvirus by real-time reverse transcription loop-mediated isothermal amplification. Microbiol. Immunol., 50(5):379-387. Yang S, Rothman RE (2004). PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect. Dis., 4(6): 337-348.