Field diagnostic assay- LAMP

1. LAMP, upcoming visual virus
diagnostic
K. Karthik,
P 1689,
VBM

2. Introduction
• Prevention and eradication of disease-starts
with diagnosis
• PCR has ruled the world of diagnosis a
while
• The need of the hour is a assay useful
at field level

3. Ideal diagnostic
1. The tests should be sensitive
2. Test should be specific
3. Reasonable cost so that the farming
community may be benefited
4. Simple protocols to perform
5. Rapidly performed
6. Adopted to any sort of climatic variation
7. Instruments used should be available
everywhere

4. Isothermal amplification Assays
1. Nucleic acid sequence-based amplification (NASBA)
2. Transcription mediated amplification (TMA)
3. Self-sustained sequence replication (3SR)
4. Strand displacement amplification (SDA)
5. Rolling circle amplification (RCA)
6. Signal mediated amplification of RNA technology
(SMART)
7. Loop-mediated isothermal amplification of DNA
(LAMP)
8. Isothermal multiple displacement amplification
(IMDA)
9. Helicase-dependent amplification (HDA)
10. Single primer isothermal amplification (SPIA)
11. Circular helicase dependent amplification (cHDA)

5. 200
180
160
140
120
100
80
60
40
20
0
Pubmed results by Year
2014 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000
Count
Year

6. LAMP
 Notomi et al. 2000, developed LAMP- auto cycling
technique
 Utilizes a polymerase with strand displacing property.
 Works under isothermal condition 60-65°C
 Results visualized directly, no post amplification
procedures needed
 Less time – 60 min. (PCR – 2-3 hr) with loop primers –
still less (Nagamine et al., 2002)
 Sensitivity – 10 to 100 times more than PCR

7. F3
C
TARGET REGIONS OF PRIMER AND QUALITIES OF LAMP PRIMER
B2 B3
0-60 bp 40-60
F2
C
F1
C
bp
F3 B2C
B3C
B1
F2 F1 B1C
BLP
FLP
5’
3’
5’
3’
FIP = F2 + F1C BIP = B2 + B1C
5’ 3’ 5’ 3’
F3
B3
BLP
FLP
OUTER PRIMERS LOOP PRIMRS
INNER PRIMERS
HPLC Purified
120-160
bp

8. Primers
• F3 and B3 -major role during strand
displacement -strand displacing primers
• FIP and BIP have their function in loop
formation
• Loop primers- shortens time and
increase sensitivity

10. Enzyme- heart of the LAMP
Bst polymerase
• Isolated from Bacillus
stearothermophilus
• Optimum temperature
for activity 63°C
• Marketed by New
England Biolabs
• Cost ~Rs. 4200 (8000
Units/ml)
Bsm polymerase
• Isolated from Bacillus
smithii
• Optimum temperature
for activity 60°C
• Marketed by
Thermoscientific
• Cost ~Rs. 3400
phi29 DNA Polymerase- Bacillus subtilis phage phi29

12. Result interpretation
• Gel electrophoresis- Ladder like pattern
• Turbidity- due to magnesium
pyrophosphate formation
• Visual- addition of dyes
• RE digestion

13. Result Visualization
S.No Agent Positive Negative
1 SYBR green Green Orange
2 Calcein Green Yellow
3 HNB Sky blue Violet
4 Propidium iodide Pink Deep red-orange

14. Stability of LAMP
• More stable compared to PCR and real time PCR
• Secure at a range of temperature, pH and a wide
range of elongation time
• Incompletely processed or non processed samples can
be used
• Cold chain is a must for PCR master mix which is not a
mandate in case of LAMP
• Taq polymerase- inhibited by urine or stools, hemin,
blood culture media, N-acetyl cystein, NaCl and
anticoagulant

15. PROPERTIES PCR LAMP
Denaturation Required for separation of strands,
enabling primer binding.
Denaturation step is not a mandate
Annealing, Extension Usually employs 3 steps as denaturation,
annealing and extension, working at
different temperature and timing
Works under a constant
temperature usually between 60-
65oC.
Time required Takes 2-3 hours based on the different
parameters.
60 min usually.
Post amplification
process
Needs agarose gel electrophoresis for
knowing the result
DNA binding dyes like SYBR green
or any metal indicator like calcein or
HNB results can be interpreted
visually
Sensitivity Can detect up to nanogram level of DNA Can detect up to femtogram level of
DNA.
Instruments Needs sophisticated instrument in order
to maintain different temperature within
a given time
Water bath can serve the purpose
DNA template
preparation
Requires template DNA preparation
which should be pure and impurities can
hinder the PCR reaction
Robust technique no need for
processing of DNA. Samples as such
can be integrated to the test.
Impurities won’t hinder the reaction
(Kaneko et al., 2007; Francois et al.,
2011)

16. LAMP for animal viruses
S.NO Pathogen Host Reference
1 West nile virus Poultry Parida et al., 2004
2 Coronavirus Ruminants Poon et al., 2005
3 Highly pathogenic avian
influenza
Poultry Imai et al., 2006; Ito
et al., 2006 ; Dinh et
al., 2011
4 FMD Ruminants Dukes et al., 2006
5 Classical swine fever Pigs Chakraborty and
Choudhury, 2012
6 Porcine cytomegalo virus Pigs Yang et al., 2012
7 Camel pox (C18L gene) Camel Venkatesan et al., 2012
8 Capripox Goats Das et al., 2012
9 Porcine circo virus 2 Pigs Zhou et al., 2011
10 ND Poultry Kirunda et al., 2012
11 IBD Poultry Xue et al., 2009
12 MD Poultry Angamuthu et al.,
2012
13 PPR (N gene) S.
Ruminants
Chandrakant et al.,
2012

17. Versions of LAMP
• RealAmp- Real time monitoring
• LAMP- LFA
• LAMP with gel packs
• Microfluidic LAMP

18. Odds of LAMP
• Product cross contamination- Cause ?
• LAMP product is so firm that it is not
degraded easily and chance of carry
over contamination exist
• Closed tube LAMP reaction is the
solution- metal indicators at the start

19. Solutions
• Tin foil method (Hong et al., 2012)
• SYBR green at cap + liquid paraffin
• Microcapsule Wax dye capsule
• 3 lab technique (Notomi et al., 2000)
• Mastermix preparation in bulk
(Angamuthu et al., 2012)

20. Conclusion
• LAMP has the advantages of sensitivity,
specificity, rapidity
• A simple test – simple result
interpretation
• Problem of cross contamination needs to
be addressed
• Lyophilized mixtures can serve as a cure
for field level diagnosis of pathogens
Eiken Chemical Company (Eiken), Tokyo, Japan