تکثیر هم‌دمایی به واسطه حلقه: روش تشخیص سریع و کم هزینه عوامل عفونی
محورهای موضوعی : میکروب شناسی تشخیصی
اعظم عسکری
1
,
محمد کارگر
2
,
صادق قربانی دالینی
3
,
عباس دوستی
4
1 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
2 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
3 - دانشگاه آزاد اسلامی، واحد جهرم، باشگاه پژوهشگران جوان
4 - دانشگاه آزاد اسلامی، واحد شهرکرد، مرکز تحقیقات بیوتکنولوژی
کلید واژه: بهینه سازی, LAMP, طراحی پرایمر,
چکیده مقاله :
روش تکثیر هم دمایی به واسطه حلقه (LAMP) مبتنی بر تکثیر اسید نوکلئیک در دمای ثابت ˚C 65-62 است. در این روش بدون نیاز به مرحله واسرشت شدن حرارتی، به کمک آنزیم DNA پلی مراز Bst به صورت هم زمان امکان جداسازی و تکثیر دو رشته DNA وجود دارد. بنابراین بر خلاف سایر روش های متداول تکثیری ، بدون نیاز به چرخه های دمایی و دستگاه ترموسایکلر و با استفاده از تجهیزات ارزان قیمتی مانند حمام آب گرم و یا بلوک حرارتی قابل انجام است. برای طراحی واکنش LAMP نیاز به 6 عدد پرایمر وجود دارد که توانایی اتصال کاملاً اختصاصی به 8 ناحیه مجزا از توالی هدف را داشته باشند. در نتیجه واکنش LAMP مقدار بسیار زیادی محصول ایجاد می شود که بدون نیاز به روش پر زحمت الکتروفورز در ژل، به کمک روش های ساده تری مانند مشاهده کدورت و یا تغییر رنگ ناشی از رنگ های فلورسنس در مخلوط واکنش به سادگی قابل تشخیص می باشد. بدین ترتیب روش LAMP با توجه به مزایایی مانند حساسیت و ویژگی مناسب ، کارایی بالا ، عدم نیاز به تجهیزات آزمایشگاهی گران قیمت و سادگی تشخیص، می تواند به عنوان ابزاری ارزشمند برای تشخیص سریع بیماری های عفونی در آزمایشگاه های کلینیکی و بیمارستانی به ویژه در کشورهای درحال توسعه به کار برده شود. این مقاله با هدف معرفی مبانی و کاربردهای روش LAMP در تشخیص عوامل عفونی تدوین گردیده است.
Loop-mediated isothermal amplification (LAMP) is nucleic acid amplification method that amplifies target isothermally at 62-65˚C. In this approach, withouth thermal denaturation step, double stranded DNA is simultaneousely denatured and synthesized by using the Bst (Bacillus stearothrmophilus) DNA polymerase with high strand displacement activity. Thus the reaction can be conducted with a cost-effective quipment such as water bath or heating block, and the thermal-cycling needs of a PCR are avoided. The LAMP reaction uses six primers that specifically recognize eight distinct regions on the target sequence. Moreover, the LAMP reaction produces a large amount of amplified products, resulting in easier detection, such as visual judgment based on the turbidity or colour change result from fluorescent dsDNA intercalating dye in the reaction mixture, so gel electrophoresis is not required. Therefore, the LAMP assay has the advantages of high specificity, sensitivity, amplification efficiency and simple detection, that without the need for expensive equipment could be applicable as valuable tool for rapid diagnosis of infectious diseases in both clinical and hospital laboratories of developing countries. The aim of this article is to introduce the principles and applications of LAMP method for detection of infectious agents.
1. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymol. 1987; 155: 335-350.
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985; 230: 1350-1354.
3. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988; 239: 487-491.
4. Compton J. Nucleic acid sequence-based amplification. Nature. 1991; 350: 91-92.
5. Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. PNAS. 1990; 87: 1874-1878.
6. Walker GT, Little MC, Nadeau JG, Shank DD. Strand displacement amplification an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 1992; 20: 1691-1696.
7. Dong J, Olano JP, McBride JW, Walker DH. Emerging pathogens: challenges and successes of molecular diagnostics. J Mol Diagn. 2008; 10:185-197.
8. Vrana KE. Advancing technologes in gene amplification. Trends in biotechnol. 1996; 14(11): 413-415.
9. Chan AB, Fox JD. NASBA and other transcription-based amplification methods for research and diagnostic microbiology. Rev Med Microbiol. 1999; 10: 185-196.
10. Gill P, Ghaemi A. Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic acids. 2008; 27 (3): 224-243.
11. Ratcliff RM, Chang G, Kok T, Sloots TP. Molecular diagnosis of medical viruses. Curr Issues Mol Biol. 2007; 9: 87-102.
12. Ihira M, Akimoto S, Miyake F, Fujita A, Sugata K, Suga S, Ohashi M, Nishimura N, Ozaki T, Asano Y, Yoshikawa T. Direct detection of human herpesvirus 6 DNA in serum by the loop-mediated isothermal amplification method. J Clin Virol 2007; 39(1): 22-26.
13. Espy MJ, Uhl JR, Sloan L, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt JE, Cockerill FR 3rd, Smith TF. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev. 2006; 19: 165-256.
14. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T, Loop mediated isothermal amplification of DNA. Nucleic Acids Res. 2000; 28(12): e63.
15. Eiken GENOME SITE. http://loopamp.eiken.co.jp/e/
16. Parida M, Sannarangaiah S, Dash PK, Rao PVL, Morita K. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol. 2008; 18(6): 407-421.
17. Tomita N, Mori Y, Kanda H, Notomi T. Loop mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protocols. 2008; 3: 877-882.
18. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop mediated isothermal amplifi cation reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun. 2001; 289:150-154.
19. Mori Y, Kiato M, Tomitam N, Notomi T. Real-Time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods. 2004; 59: 145-157.
20. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loop mediated isothermal amplification reaction using a non denaturated template. Clin Chem. 2001; 47: 1742-1743.
21. Kaneko H, Iida T, Aoki K. Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification. J Clin Microbiol. 2005; 43(7): 3290-3296.
22. Kaneko H, Kawana T, Fukushima E, Suzutani T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods. 2007; 70: 499-501.
23. Whiting, SH, Champoux JJ. Properties of strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase: mechanistic implications. J Mol Biol. 1998; 278: 559-577.
24. Nagamine K, Kuzuhara Y, Notomi T. Isolation of single-stranded DNA from loop-mediated isothermal amplification products. Biochem Biophys Res Commun. 2002; 290: 1250-4 to1195-8.
25. Kargar M, Askari A, Doosti A, Ghorbani-Dalini S. Comparison of efficiency of ELISA, LAMP and nested-PCR assays for detection of Hepatitis C Virus. Journal of Isfahan Medical School. 2013; [In Press].
26. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002; 16: 223-229.
27. New England Biolabs Company. Bst DNA Polymerase Lg Frag from New England Biolabs. http://www.neb.com.
28. Aviel RonenS, Qi Zhu C. Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues. BMC Genomics. 2006; 7: 312.
29. Hafner GJ, Yang IC, Wolter LC, Stafford MR, Giffard PM. Isothermal amplification and multimerization of DNA by Bst DNA polymerase. Biotechniques.2001; 30: 852-867.
30. Rees WA, Rees WA, Yager TD, Korte J, von Hippel PH. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 1993; 32: 137-144.
31. Henke W, Herdel K, Jung K, Schnorr DA, Loening S. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 1997; 25: 3957-3958.
32. Rajendrakumar CS, Suryanarayana T, Reddy AR. DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process. FEBS Letters. 1997; 410: 201-205.
33. Karleson F, Steen H, Nesland J. SYBR green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction. J Virol methods. 1995: 55: 153-156.
34. Zipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic acids Res. 2004; 32 (12): e103.
35. Hill J, Beriwal S, Chandra I, Paul VK, Kapil A, Singh T Wadowsky RM, Singh V, Goyal A, Jahnukainen T, Johnson JR, Tarr PI, Vats A. Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. J Clin Microbiol. 2008; 46: 2800-2804.
36. Bista B, Ishwad CM, Wadowsky R. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus. J Clin Microbiol. 2007; 1581-1587.
37. Pham HM, Nakajima C, Ohashi K, Onuma M. Loop mediated isothermal amplification for rapid detection of Newcastle disease virus. J Clin Microbiol. 2005; 43(4): 1646-1650.
38. Parida M, Horioke K, Ishida H, Dash PK, Saxena P, Jana AM, Islam MA, Inoue S, Hosaka N, Morita K. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcriptionloop- mediated isothermal amplification assay. J Clin Microbiol. 2005; 43: 2895-2903.
39. Kargar M, Askari A, Doosti A, Ghorbani-Dalini S. Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus. Indian J Virol. 2012; 23(1): 18-23.
40. Shahhosseiny MH, Esmailii Z, Moslemi E, Yaghmaii P. Loop mediated isothermal amplification of hepatitis B virus in hemodialysis patients. Journal of Microbial World. 2009; 2(3): 183-192. [In Persian]
41. Imai M, Ninomiya A, Minekawa H, Notomi T, Ischzaki T, Van Tu P, Tien NT, Tashiro M, Odagiri T. Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method. J Virol Methods. 2007; 141: 173-180.
42. Yoda T, Suzuki Y, Yamazaki K, Sakon N, Aoyama I, Tsukamoto T. Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses. J Med Virol. 2007; 79: 326-334.
43. Parida M, Santhosh SR, Dash PK. Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus. J Clin Microbiol. 2006; 44(11): 4172-4178.
44. Parida M, Guillermo P, Shingo I, Inoue S, Hasebe F, Morita K. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus. J Clin Microbiol. 2004; 42(1): 257-263.
45. Toriniwa H, Komiya T. Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loopmediated isothermal amplification. Microbiol Immunol. 2006; 50: 379-387.
46. Hagiwara M, Sasaki H, Matsuo K, Honda M, Kawase M, Nakagawa H. Loop mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18. J Med Virol. 2007; 79(5): 605-615.
47. Fukuda S, Takao S, Kuwayama M, Shimazu Y, Miyazaki K. Rapid detection of norovirus from fecal specimens by real-time reverse transcription–loop-mediated isothermal amplification assay. J Clin Microbiol. 2006; 44(4): 1376-1381.
48. Parida M. Rapid and real-time detection technologies for emerging viruses of biomedical importance. J Biosci. 2008; 33(4): 617-628.
49. Mori Y, Hirano T, Notomi T. Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol. 2006; 6(3): 1-10.
50. Okafuji T, Yoshida N, Fujino M, Motegi Y Ihara T, Ota Y, Notomi T, Nakayama T. Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification. J Clin Microbiol. 2005; 43(4): 1625-1631.
51. Mori N, Motegi Y, Shimamura Y, Ezaki T, Natsumeda T, Yonekawa T, Ota Y, Notomi T, Nakayama T. Development of a new method for diagnosis of rubella virus infection by reverse transcription–loop-mediated isothermal amplification. J Clin Microbial. 2006; 44(9): 3268-3273.
52. Askari A, Kargar M, Doosti A. 2011. Comparative evaluation of LAMP, Nested PCR and EIA methods for diagnosis of Hepatitis C Virus. 1st International and 12th Iranian Congress of Microbiology, Kermanshah, Iran. No.1427
53. Alhassan A, Govind Y, Thanh Tam N. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res. 2007; 100: 1165-1168.
54. Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, Hasebe F, Morita K. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol. 2004; 42(5): 1956-1961.
55. Lan X, Yang B, Li BY, Yin XP, Li XR, Liu JX. Reverse transcription–loop-mediated isothermal amplification assay for rapid detection of hepatitis E virus. J Clin Microbiol. 2009; 47(7): 2304-2306.
56. Iwamoto Y, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellular in sputum samples. J Clin Microbial. 2003; 41: 2616-22.
57. Goto M, Hayashidani H, Takatori K, Kudo, YH. Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay. Lett Appl Microbiol. 2007; 45: 100-107.
58. Ohtsuki R, Kawamoto K, Kato Y, Shah MM, Ezaki T, Makino SI. Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method. J Appl Microbiol. 2008; 104: 1815-1823.
59. Yamazaki W, Taguchi M, Ishibashi M, Kitazato M, Nukina M, Misawa N, Inoue K. Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. J Med Microbial. 2008; 57: 444-451.
60. Annaka T. Rapid and simple detection of Lgionella species by LAMP, a new DNA amplification method. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi. 2003; 14: 5660-5662.
61. Ohatsuka K, Yanagawa K, Takatori K, Hara-Kudo Y. Detection of Salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates. Apple Environ Microbial. 2005; 71: 6730-5.
62. Hara-Kudo Y, Nemoto J, Ohtsuka K, ayoko Ohtsuka K, Segawa Y, Takatori K, Kojima T, Ikedo M. Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J Med Microbiol. 2007; 56: 398-406.
63. Zhao X, Li Y, Wang L, You L, Xu Z, Li L, He X, Liu Y, Wang J, Yang L. Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples. Mol Biol Rep. 2010; 37(5): 2183-2188
64. Maruyama F, Kenzaka T, Yamaguchi N, Tani K, Nasu M. Detection of bacteria carrying the stx2 gene by in situ loop-mediated isothermal amplification. Appl Environ Microbiol. 2003; 69(8): 5023-5028.
65. Enomoto Y, Tetsushi Y, Ihira M, Akimoto S, Miyake F, Usui C, Suga S, Suzuki K, Kawana T, Nishiyama Y, Asano Y. Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method. J Clin Microbiol. 2005; 43(2): 951-955.
66. Ihira M, Yoshikawa T, Enomoto Y, Akimoto S, Ohashi M, Suga S, Nishimura N, Ozaki T, Nishiyama Y, Notomi T, Ohta Y, Asano Y. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA Amplification method, loop-mediated isothermal amplification. J Clin Microbial. 2004; 42(1): 140-145.
67. Suzuki R, Yoshikawa T, Ihira M, Enomoto Y, Inagaki S, Matsumoto K, Kato K, Kudo K, Kojima S, Asano Y. Development of the loop-mediated isothermal amplification method for rapid detection of cytomegalovirus DNA. J Virol Methods. 2006; 132(1–2): 216-221.
68. Jayavardena S, Cheung CY, Barr I, Chan KH, Chen H, Guan Y, Peiris JS, Poon LL. Loop-mediated isothermal amplification for influenza A (H5N1) virus. Emerg Infect Dis. 2007; 13(6): 899-901.
69. Curtis KA, Rudolph DL, Owen SM. Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP). J Virol Methods. 2008; 151: 264-270.
70. Yoneyama T, Kiyohara T, Shimasaki N, Kobayashi G, Ota Y, Notomi T, Totsuka A, Wakita T. Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay. J Virol Methods. 2007; 145(2): 162-168.
71. Paris HD, Imwong MM, Faiz A. Loop-mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria. Am J Trop Med Hyg. 2007; 77(5): 972-976.
72. Shahhosseiny MH, Rezaei Amirabadi A, Yousefi J, Ghahri M, Azadmanesh K, Moslemi E. LAMP detection of Cryptococcus neoformans in AIDS patients. Journal of Microbial World. 2010; 2(4): 235-242. [In Persian].
73. Iwasaki M, Yonekawa T, Otuska K, Suzuki W, Nagamin K, Hase K, Horigome T, Notomi T, Kanda H. Validation of the loop-mediated isothermal amplification method for single nucleotide polymorphism genotyping with whole blood. Genome Lett. 2003; 2: 119-126.
74. Nakamura N, Ito K, Takahashi M, Hashimoto K, Kawamoto M, Yamanaka M, Taniguchi A, Kamatani N, Gemma N. Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip. Anal Chem. 2007; 79(24): 9484-9493.
75. Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, Okada M, Moriya K, Koike K. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect Chemother. 2007; 13(3): 134-140.
76. Sekiguchi J, Asagi T, Akiyama TM, Kasai A, Mizuguchi Y, Araake M, Fujino T, Kikuchi H, Sasaki S, Watari H, Kojima T, Miki H, Kanemitsu K, Kunishima H, Kikuchi Y, Kaku M, Yoshikura H, Kuratsuji T, Kirikae T. Outbreaks of multi drug resistant Pseudomonas aeruginosa in community hospitals in Japan. J Clin Microbiol. 2007; 45: 979-989.
77. Liu W, Zou D, Li Y, Wang X, He X, Wei X, Shao C, Li X, Shang W, Yu K, Liu D, Li Y, Guo J, Yin Z, Yuan J. Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification. J Clin Microbiol. 2012; 50(5):1580-5. doi: 10.1128/JCM.06647-11.