Njiru, 2012 has described that " Lack of effective point of care diagnostic tests applicable in resource-poor endemic areas is a critical barrier to effective treatment and control of infectious diseases.” Therefore, Innovations in biotechnology that combine molecular biology, microfabrication and bioinformatics are moving nucleic acid technologies from futuristic possibilities to common laboratory techniques and modes for diagnoses. In this context, LAMP (Loop Mediated Isothermal Amplification) is a highly sensitive and specific DNA/RNA amplification method. Advantage of LAMP is isothermal reaction condition and therefore, LAMP is affordable because of no need to have expensive thermal cycler.

2. (Image 1 adapted from The African Executive: The West Riding on Africa’s Misery, http://www.africanexecutive.com
Image 2 Courtesy Dr. R. K.Sharma, CIRB, Hisar)
“Problems: third world and developing Nations face”

3. (http://www.medicine.cmu.ac.th/dept/parasite/
nematode/wbmf.htm)
The Invaders . . .
Bacteria
Viruses
Fungi
Protista
Worms
(http://www.cdc.gov/mrsa/mrsa_initiative/skin_inf
ection/mrsa_photo_9994.html)
MRSA
HIV
(http://www.scientificamerican.com/article.cfm?id
=experts-where-did-viruses-come-fr)
W. bancroftiT. brucei
(© 2011, American Society for Microbiology)

4. Each year in sub-Saharan Africa, ∼12 million
people die……
animals too…..
Infectious diseases-HIV infection, malaria, and
tuberculosis….. endemic, epidemic & pandemic
Quality laboratory testing is crucial
need to promote rational, cost-effective diagnostic
methods
(Petti et al., 2006)

5. “Lack of effective point of care diagnostic
tests applicable in resource-poor endemic
areas is a critical barrier to effective
treatment and control of infectious diseases”
(Njiru , 2012)
“Diagnosis is important not only for the
prescribing of effective drugs for appropriate
patients in adequate doses but also for
preventing the evolution of resistant
microorganisms”
(Mori and Notomi, 2009)

6. Common Diagnostic Methods
Clinical signs
Animal inoculation
Smears
CMT
…….
Ab and Ag detection (ELISA…)
In vitro culture
DNA hybridization
PCR
…….
Field Methods
Laboratory Methods
Easy-to-Use
Affordable
Unspecific clinical signs
Low specificity
Low sensitivity
High specificity
High sensitivity
Complicated
Expensive
Time consuming

7. The Need…………..
WHO recommends that an ideal diagnostic test suitable for
developing, underdeveloped and undeveloped countries
should be
“ASSURED”
☻Affordable
☻ Sensitive
☻ Specific
☻ User-friendly
☻ Robust and rapid
☻ Equipment free
☻Deliverable to the end user
(Mabey et al.,2004)

8. ♞Polymerase chain reaction (PCR)
♞Ligase chain reaction (LCR)
♞Nucleic acid sequence-based
amplification (NASBA)
♞Strand Displacement
Amplification (SDA)
“Introduced into routine diagnostics as nucleic acid
amplification tests (NATs)”
Amplification methods like…………..
commercialized as kits
(Versalovic et al.,2002)

9. LAMP (Loop mediated isothermal amplification)
Originally reported by Notomi et al in 2000 of EIKEN Chemical
Co. Ltd., Japan
(http://www.eiken.co.jp/en/)
As of 17th April 2013, PubMed database has listed more than
820 articles on this topic

10. Bst DNA polymerase with strand displacement
activity at 65℃
The whole amplification to 109 – 1010 copies is
finished within 15 to 60 min at 65℃, isothermally
Amplification and detection of gene can be
completed in a single step
No need for a step to denature double stranded
into a single stranded form

11. The amplification efficiency is extremely high
Reduced total cost- not require special reagents or
sophisticated equipments
Amplified products have a structure consisting of
alternately inverted repeats of the target sequence
on the same strand
Amplification can be done with RNA templates
following the same procedure as with DNA
templates, simply through the addition of reverse
transcriptase (RT-LAMP)

12. Design of primers
4 types of primers based on the 6 distinct regions of
the target gene: the F3c, F2c and F1c regions at the
3' side and the B1, B2 and B3 regions at the 5' side

13. FIP Forward Inner Primer (FIP) consists of the F2
region (at the 3' end) that is complementary to
the F2c region, and the same sequence as the
F1c region at the 5' end
F3
Primer
Forward Outer Primer consists of the F3 region
that is complementary to the F3c region
BIP Backward Inner Primer (BIP) consists of the B2
region (at the 3' end) that is complementary to
the B2c region, and the same sequence as the
B1c region at the 5' end
B3
Primer
Backward Outer Primer consists of the B3
region that is complementary to the B3c region

14. Loop Mediated Isothermal Amplification
Primer Regions
( Parida et al., 2008)
Two loop primers - forward loop primer (FLP) and backward loop primer (BLP)
accelerate the amplification reaction by binding to additional sites that are not
accessed by internal primers

15. Softwares…….
(www.premierbiosoft.com/isot
hermal/lamp.html)
(http://loopamp.eiken.co.jp/e/lamp/primer.html)
(Torres et al. BMC Bioinformatics , 2011)
(Salinas and Little, 2012)

16. PRINCIPLE OF LAMP
(A) Non-Cyclic Steps : generation of stem loop
DNA with dumbbell-shaped structure at both
ends

17. One of the LAMP primers anneal to the complimentary
sequence of double stranded target DNA
Initiates DNA synthesis using the DNA polymerase with
strand displacement activity, displacing and releasing a
single stranded DNA
Unlike PCR, no need for heat denaturation of ds DNA
STEP 1

18. Through the activity of DNA polymerase with strand
displacement activity, a DNA strand complementary
to the template DNA is synthesized, starting from
the 3' end of the F2 region of the FIP
STEP 2

19. The F3 Primer anneals to the F3c region, outside of
FIP, on the target DNA and
Initiates strand displacement DNA synthesis,
releasing the FIP-linked complementary strand
STEP 3

20. A double strand is formed from the DNA strand
synthesized from the F3 Primer and the template
DNA strand
STEP 4

21. The FIP-linked complementary strand is released as
a single strand because of the displacement by the
DNA strand synthesized from the F3 Primer
Released single strand forms a stem-loop structure
at the 5' end because of the complementary F1c
and F1 regions
STEP 5

22. This single strand DNA in Step (5) serves as a template
for BIP-initiated DNA synthesis and subsequent B3-
primed strand displacement DNA synthesis
STEP 6

23. Double stranded DNA is produced through the
processes described in Step (6)
STEP 7

24. The BIP-linked complementary strand displaced in
Step (6) forms a structure with stem-loops at each
end, which looks like a dumbbell structure
This structure serves as the starting structure for
the amplification cycle in the LAMP method (LAMP
cycling)
STEP 8

25. (A) Non-Cyclic Step : generation of stem loop
DNA with dumbbell-shaped structure at both
ends that is ready to enter into cyclic
amplification step

26. (B) Cycling amplification step

27. Using Loop Primers

28. Time saving by Loop Primer……
Time required for amplification with Loop Primers is one-
third to one-half of that without Loop Primer
With the use of Loop Primers, amplification can be
achieved within 30 minutes
Original method:
no Loop Primer
Rapid method:
with Loop Primers

29. LAMP does not require thermal cycler
• Cycle reaction
• Denature (95℃)
• Annealing (50~60℃)
• Polymerization (72℃)
• ~ >1 hour
• Isothermal reaction
• 60- 65℃
• Max 1 hour
PCR LAMP

30. Detection
Visually
1.Fluorescence – Eye
2.Turbidity – Turbidmeter (or Eye)
Gel electrophoresis

31. LAMP product is visible to naked eyes
(DNA)n-1+dNTP → (DNA)n + P2O7
4-
P2O7
4- + 2Mg2+ → Mg2P2O7 ↓
PPT
{Biochemical and Biophysical Research Communications 289, 150–154 (2001)}
Insoluble Turbid

32. Amplification products are stem-loop DNA structures with
several inverted repeats of the target and cauliflower-like
structures with multiple loops, yielding ~ >500 mg/mL (10-
20 μg/25 μl)
An increase in the turbidity of the reaction mixture
according to the production of precipitate correlates with
the amount of DNA synthesized
( Mori et al., 2001)

33. Turbidity increases - when concentration of
pyrophosphate ion exceeded 0.5 mM
on a 25-μl scale reaction, a DNA yield of more
than 4 μg is required to elevate the
pyrophosphate ion concentration to > 0.5 mM
LAMP reaction synthesizes 10 μg/25 μl or more
DNA, and therefore produced pyrophosphate ion
react with magnesium ion to induce the
precipitate
DNA yield by PCR is about 0.2 μg/25 μl and the
resulting pyrophosphate ion approximates 0.02
mM ----------> No precipitate

34. Nature Protocols 3, 877 - 882 (2008)
Detection using a fluorescent metal indicator (calcein)
Green fluorescence
by free calcein

35. (a) Irradiating the tube using a handheld-UV lamp
(wavelength: 365 nm) from the bottom
(b) Under daylight. Plus sign denotes positive reaction
(with target DNA), minus sign denotes negative
reaction (without target DNA)
(Tomita et al., 2008)

36. (a) HNB: The color changes from violet
(negative reaction) to sky blue
(positive reaction)
(b) SYBR green and
(c) Calcein
The color changes from orange
(negative reaction) to yellow
(positive reaction)
Detection of 10-fold serially diluted λ DNA using
Hydroxy Naphthol Blue (HNB), Syber Green and Calcein
(Goto et al., 2009)

37. Detection of 10-fold serially diluted λ DNA using
Hydroxy Naphthol Blue (HNB), Syber Green and Calcein
Visualization under UV irradiation
(b) SYBR green and
(c)Calcein
Bright fluorescence indicates a
positive reaction
• Tube 1, 1:103 dilution
(1.58 × 107 copies/tube)
• tube 2, 1:104 dilution
• tube 3, 1:105 dilution
• tube 4, 1:106 dilution
• tube 5, 1:107 dilution
• tube 6, 1:108 dilution
• tube 7, 1:109 dilution
• tube 8, no template.
(Goto et al., 2009)

38. Loopamp Realtime Turbidimeter (LA-500)
With the time and temperature preset, gene
amplification will occur, and the detection can be done
by simultaneously monitoring the white turbidity
caused by the existence of magnesium
pyrophosphate, the amplification by-products

39. Analysis of the loop-mediated isothermal
amplification (LAMP) reaction products using
agarose gel electrophoresis (Tomita et al., 2008)

40. (LaBarre et al.,2011)
(A) assembled incubator, (B) incubator lid with built-in spring timer, (C) CaO chamber
(w/ CaO added), (D) assay tubes, and (E) thermocouple wires
Simple, Inexpensive Device for Nucleic Acid
Amplification without Electricity
Fifth-generation prototype design made from
a reusable $4 insulated soup thermos
container

41. (Curtis et al.,2012)
Non-instrumented nucleic acid (NINA) heaters with DaqPRO 5300 Data recorder
Isothermal Amplification Using a Chemical
Heating Device

42. LAMP Vs PCR
• LAMP does not require an expensive thermocycler
• Amplification specificity is extremely high as LAMP
requires 4/6 oligonucleotide primers that recognize 6/8
distinct regions on the target DNA
• Detection limit : LAMP ≥ PCR
• Detection time : LAMP < PCR
• LAMP reaction: accelerated by two loop primers
• Visualization of DNA products by LAMP:
(a) Eye – turbidity, colour change
(b) Real Time Turbidimeter
(C) Electrophoresis
(Goto et al., 2009)

43. LAMP can be done by rather crude DNA sample
PCR is susceptible to hemoglobin, Ig and Heparin
LAMP resists contamination of above mentioned
materials
LAMP can amplify parasite DNA from fresh
infected blood
It means that LAMP can be done by using rather
crude DNA extracted by simple methods

44. Practical applications

45. • As of April17, 2013, PubMed database has listed
more than 820 articles on this topic
• Before 2008, the Severe Acute Respiratory
Syndrome (SARS) coronavirus detection kit was
the only LAMP reagent approved for in vitro
diagnostics (IVD) in Japan
• Currently, the number of approved LAMP
reagents in Japan has increased to eight:
SARS coronavirus, Mycobacterium tuberculosis
(TB), Mycoplasma pneumoniae, Legionella ,
influenza type A virus, H1 pdm 2009 influenza
virus, H5 influenza virus, and human papilloma
virus (HPV)

46. LAMP - TB for the peripheral level
• LAMP reagent kit for detecting the M. tuberculosis
complex (Loopamp MTBC detection kit, TB-LAMP;
Eiken Chemical, Tokyo, Japan) was launched in April
2011
• The test process has been made faster and simpler;
by using the kit named the Loopamp PURE DNA
extraction kit (Eiken Chemical) for sputum
processing
• TB-LAMP is provided as a dry reagent, allowing
easier storage, that is, it can be stored at room
temperature with satisfactory shelf life

47. Collaborative development with FIND
(Foundation for Innovative New Diagnostics)
FIND: non-profit foundation for developing diagnostic tools for poverty-related diseases

48. Overview of PURE Method
(Procedure for Ultra Rapid Extraction)
( Okamoto, 2011)

49. PURE-TB LAMP Operation
( Okamoto, 2011)

50. LAMP Reagents in Dried Form
Dried LAMP reagents fixed
on the lids
↓
Reconstitute by PURE
treated sample solutions
Save steps for preparing LAMP reaction solutions
Stable at room temperature for at least one year
（no need to store in the fridge）
( adapted from Okamoto, 2011)

51. • Malaria LAMP kit (Loopamp MALARIA Pan/Pf
detection kit) has been launched (diagnostic
accuracy of the kit is similar to that of nested PCR
with greatly reduced time to availability of results)
• Loopamp Trypanosoma brucei detection kit---
implications for Surra (on the basis of its reactivity
with Trypanosoma evansi and Trypanosoma
equiperdum)
• A LAMP assay system for detecting Clostridium
difficile (illumigene C. difficile; Meridian
Bioscience, Cincinnati, OH, USA) has been
developed in the United States

52. Thailand: Cholera outbreaks in Tak province, 2008
(Okamoto, 2011)

53. • Application of LAMP to the world’s three major
diseases - TB, malaria, and human
immunodeficiency virus (HIV), has also been
aggressively advanced since 2009, and more than
40 scientific articles on these diseases have been
published
• Of 17 NTDs recognized by WHO , 14 were studied
using LAMP-
dengue, rabies, buruli ulcer, leprosy, Chagas
disease, human African trypanosomiasis,
leishmaniasis, cysticercosis, echinococcosis,
foodborne trematode infections, lymphatic
filariasis, schistosomiasis, soil-transmitted
helminthiases, and yaws

54. Magnetic bead-based assay for MRSA rapid detection –
what an attractive idea!
(Wang et al., 2011, Lab Chip )

55. A proposed three-step LAMP
method for diagnosis of
neglected tropical diseases
(Njiru, 2012)

57. MALE FEMALE
The natural light observation of the reaction tube
using digital camera: Colour change after addition
of EtBr (1mM, 1:5 v/v) ( left shows the positive
reaction (male) and right the negative reaction
(female))
The tube with salmon pink coloration
was judged as a positive reaction
(male), the tube with wine coloration
was judged as a negative reaction
(female)
(Zoheir and Allam, 2011)

58. MALE FEMALE
The natural light observation of the reaction tube
using digital camera: Precipitate formation after
addition of 5 μl of CuSO4 (3M)– left shows the
positive reaction (male) and right the negative
reaction (female)
(Zoheir and Allam, 2011)
The tubes with a deposition were
identified as negative LAMP
reaction (female), while the others
without obvious one were active or
positive LAMP reaction (male)

59. In India………..

61. LAMP is highly sensitive and specific DNA/RNA amplification
method
Advantage of LAMP is isothermal reaction condition, hereby
LAMP is affordable because of no need to have expensive
thermal cycler
Although recommended reagent storage temperature is -
20oC, reagents can be stored at ambient temperature for at
least 2 weeks. Hereby there is no need to have cold chain
for reagent distribution
Crude DNA preparation can be used as LAMP template DNA.
Cost of LAMP can be reduced to approximately 1 USD/test
or cheaper
LAMP can be a field molecular diagnostic method for
infectious diseases……….food inspection………environmental
testing……….. Sexing……….and so on….
Conclusions and Perspective

62. “Innovations in biotechnology that combine molecular
biology, microfabrication and bioinformatics are
moving nucleic acid technologies from futuristic
possibilities to common laboratory techniques and
modes for diagnoses.”

64. THANK
YOU