LAMP, upcoming visual virus diagnostic

K. Karthik,

P 1689,

VBM

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

Ideal diagnostic

1. The tests should be sensitive2. Test should be specific3. 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

Isothermal amplification Assays1. 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)

0

20

40

60

80

100

120

140

160

180

200

2014 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000

Co

un

t

Year

Pubmed results by Year

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

F3C

F3 B3C

B3B2

B2C

B1

B1CF1F2

F2C

F1C

BLP

FLP

5’

5’

3’

3’

FIP = F2 + F1C BIP = B2 + B1C

5’ 5’3’ 3’

B3

F3

FLP

BLP

LOOP PRIMRSOUTER PRIMERSINNER PRIMERS

HPLC Purified

0-60 bp 40-60 bp

120-160 bp

TARGET REGIONS OF PRIMER AND QUALITIES OF LAMP PRIMER

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

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

Result interpretation

• Gel electrophoresis- Ladder like pattern

• Turbidity- due to magnesium pyrophosphate formation

• Visual- addition of dyes

• RE digestion

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

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

PROPERTIES PCR LAMPDenaturation 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)

LAMP for animal virusesS.NO Pathogen Host Reference1 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

Versions of LAMP

• RealAmp- Real time monitoring

• LAMP- LFA

• LAMP with gel packs

• Microfluidic LAMP

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

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)

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