Loop Mediated Isothermal Amplification (LAMP)
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The document presents a detailed overview of loop-mediated isothermal amplification (LAMP) and its application in detecting the tomato yellow leaf curl virus (TYLCV). It contrasts LAMP with traditional PCR technology, highlighting advantages such as higher specificity and efficiency, and demonstrates the effectiveness of LAMP in amplifying viral DNA from infected tomato plants. The study includes methodologies, results of amplification tests, and discussions on LAMP's significance in diagnosing viral infections.
Loop Mediated Isothermal Amplification (LAMP)
- 1. A PRESENTATION ON Loop-Mediated Isothermal Amplification Presented by MD ROBEL AHMED STUDENT ID: L20192E020101 1ST YEAR 1ST SEMESTER FACULTY OF LIFE SCIENCE AND MECICINE Presented to ZHIYOU DU PROFESSOR , FACULTY OF LIFE SCIENCE AND MEDICINE ZHEJIANG SCI-TECH UNIVERSITY DATE OF SUBMISSION: 27th SEPTEMBER 2019
- 2. Outlines What is Loop Mediated Isothermal Amplification? Differences of LAMP with conventional PCR technology. Application of LAMP in TY LCV detection assay.
- 4. 3’ F3c F2c F1c B1 B2 B3 5’ F1F2 B3cB2cB1cF35’ 3’ F3 Forward Outer Primer B3 Backward Outer Primer F2 Forward Inner Primer B2 Backward Inner Primer LAMP Experiment Design
- 5. How Lamp Works? 3’ F3c F2c F1c B1 B2 B3 5’ Denaturation separates two strands
- 6. How Lamp Works? 3’ F3c F2c F1c B1 B2 B3 5’ 3’ F3c F2c F1c B1 B2 B3 5’ F3 F2 FIP
- 7. F1F2 B3cB2cB1cF35’ 3’ B2 Backward Inner Primer F1F2 B3cB2cB1cF35’ 3’ B3 How Lamp Works?
- 10. LAMP vs. PCR Isothermal Reaction Cyclic Reaction Doesn’t require expensive thermo- cycler Require thermo-cycler Detection limit is greater Detection limit is lower Amplification specificity is higher as uses 4/6 oligonucleotides or more Amplification specificity is lower than LAMP Visualization DNA could be done through eyes, electrophoresis, turbidimeter. Visualization of DNA is done through Gel electrophoresis.
- 11. Application of LAMP in TY LCV detection assay Detection of Tomato Yellow Leaf Curl Virus by Loop Mediated Isothermal Amplification reaction (TYLCV) Abstract Whitefly transmitted geminivirus. Amplified from total DNA extract of TYLCV infected tomato plant Lycopersicon esculentum. Synthesize large amount of DNA Bi-product is pyrophosphate ion White Precipitation
- 12. Genomic Organization of VIRUS Fig. Genomic organization of TYLCV-Aichi or -Is. Open reading frames (ORFs) are shown as black arrows; V denotes ORFs on the virion strand, and C denotes ORFs on the complementary strand. IR indicates the intergenic region. Region 1 is recognized by LAMP primers SF371-SF374. Region 2 is recognized by LAMP primers SF448/SF451. Region 3 is recognized by LAMP primers SF432/ SF435. Region 4 is recognized by PCR primers SF301 and SF303.
- 13. Genomic Organization of VIRUS The sequences of three sets of primers SF371/374, SF448/451 and PCR primers were designed from the DNA sequences of TYLCV-Aichi (5). The sequences of a set of primers SF432, SF433, SF434 and SF435 were designed from the DNA sequences of TYLCV-Is
- 14. Introduction First found in Japan 1998. Causes 100 percent yield loss is all over the world. Therefore, several methods are discovered to detect the virus. Some disadvantage of conventional PCR is Rapid thermal cycling Insufficient specificity Low amplification efficiency.
- 15. LAMP procedure has overcome most of this problems and here, Isothermal condition Simple and inexpensive reaction. Easier and convenient detection method, High amplification efficiency. Rapid and faster result, This experiment uses two form of Virus, 1. TYLCV-Aichi 2. TYLCV-Nagasaki Introduction
- 16. Materials and Methods Whiteflies were reared on radish plants Covered with polypropylene sheets Moved to TYLCV-host plants Plant is infected by the VIRUS 1. TYLCV strain, Plants, insects
- 17. 2. Extraction of total DNA Uses leaf tissue sample to extract DNA DNeasy Plant Mini Kit (Qiagen) One microliter DNA extract was used in 25 ml PCR or LAMP Materials and Methods
- 18. 3. PCR based detection Primer: SF 301 and SF 303 PCR reaction component: 200 mM of each dNTPs, 0.2 mM of each primer, 2 mM MgCl2, 10 mM Tris/HCl (pH 8.3), 50 mM KCl and 2.5 units of AmpiTaq Gold DNA polymerase Total 40 cycle of reaction: denaturing for 20 s at 94 8C, annealing for40 s at 55 8C and DNA extension for 20 s at 72 8C Electrophoresis: 1 percent agarose gel in EDTA buffer (pH=8.0) Materials and Methods
- 19. 4. LAMP based detection Primer: Three sets of primers Amplification reaction: performed at 65.8 degree C by mixing 1.6 mM each of FIP and BIP primer, 0.2 mM each of F3 and B3 primer, 1.4 mM dNTPs, 8 U of Bst DNA polymerase, 0.8 M betaine (Sigma-Aldrich), 2 mM MgSO4, and 1 ml of extracted template DNA. LAMP amplification of TYLCV DNA from tomato infected with TYLCV-Aichi (A), (tubes 1, 4, 7); tomato infected with TYLCV-Nagasaki (N), (tubes 2, 5, 8); uninfected tomato (U), (tubes 3, 6, 9). Primers were SF371/374 (tubes 1, 2, 3); SF448/451 (tubes 4, 5, 6); SF432/435 (tubes 7, 8, 9). LAMP reaction tubes showing white precipitate of MgPPi when TYLCV DNA was present. Materials and Methods
- 20. Fig. LAMP amplification of TYLCV DNA by simple method of extraction from tomato infected with TYLCV-Aichi (A) (tube 1) tomato infected with TYLCV-Nagasaki (N) (tube 2) uninfected tomato (U) (tube 3). Materials and Methods
- 21. Fig. Agarose gel electrophoresis of (A) PCR and, (B) LAMP using the primers SF371/SF374 products from total DNA isolated from tomatoes tomato infected with TYLCV-Aichi (lane 1) tomato infected with TYLCV-Nagasaki (lane 2) uninfected tomato (lane 3). Lane M, DNA size marker (100 bp ladder) Materials and Methods
- 22. Result Amplification of the TYLCV genome from a TYLCV infected plant Key Points: Two sets of primers were used to detect individual strains of TYLCV SF301 and SF303, were used to amplify TYLCV genomic molecules TYLCV-Aichi and TYLCV-Nagasaki were detected with LAMP using primers SF432, SF433, SF434 and SF435 or primers SF448, SF449, SF450 and SF451, respectively 187 bp was amplified from DNA of tomatoes infected with TYLCV-Aichi and TYLCV-Nagasaki, But not from DNA of a healthy tomato. LAMP amplification was performed at 65.8C using four primers, SF371, SF372, SF373, and SF374 visible white precipitate of magnesium pyrophosphate was produced in the presence of TYLCV DNA.
- 23. Result Threshold of TYLCV detection in plants and whiteflies by LAMP Attempted to determine the threshold of TYLCV detection in plants. Extracts from TYLCV infected tomatoes were mixed with extracts from healthy tomatoes. Prepare serial 10-fold dilutions of the TYLCV-infected plant DNA. After 103 dilution, PCR signal lost, but not LAMP. Using LAMP, TYLCV DNA was amplified from plant extracts diluted up to 105.
- 24. Fig. Agarose gel electrophoresis of (A) PCR and (B) LAMP products from total DNA isolated from tomato infected with TYLCV after serial 10-fold DNA dilutions from 100 to 107 . Lane M, DNA size marker (100 bp ladder). Result
- 25. Result
- 26. Fig. Alignment of the DNA sequences of LAMP primer-recognized regions for nine isolates of TYLCV. Nucleotide sequences differing from that of the Aichi isolate are shown as lower case letters, and the identical nucleotides as periods. The sequences of primer sites were shadowed. The Nagasaki isolate sequence was determined in this laboratory (Fukuta et al., unpublished); sequences of isolates from Aichi, Israel, Spain, Portugal, Iran and Puerto Rico were obtained from GenBank with the accession numbers; AB014347, X15656, AF071228, AF105975, AJ132711 and AY134494. Result
- 27. Discussion We have found that among various detection method LAMP’s significance is very high. Advantage of high specificity high amplification easy detection Replacing gel electrophoresis to pyrophosphate precipitant. Making sensitive viral dilution is much easier(endpoint dilution in PCR 102 fold but LAMP 105−7 fold. It also shows that primer SF 371 to SF374 recognizes or matches to all the sequences found worldwide.
- 28. Discussion LAMP technology has extend the ability to diagnose viral infections in tern of plants or insects or others vector and It has been also useful to get molecular and epidemiological data and will help to predict Outbreaks of disease.
- 29. References All the Authors of this research paper and related references titled” Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction.” Journal of Virological Methods,112(2003) 35-40 Special thanks to Professor Zhiyou Du for support and Providing this opportunity to present such kinds or topic