Loop Mediated Isothermal Amplification (LAMP) is a kinds of PCR reaction. This technology is most reliable and convenient than conventional PCR procedure. We can call it updated version of PCR. Rapid, Easy detectable and cheap to accomplish the process.

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.

3. LAMP
Loop Mediated
Isothermal
Amplification

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?

8. 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