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(Image 1 adapted from The African Executive: The West Riding on Africa’s Misery, http://www.africanexecutive.com
Image 2 C...
(http://www.medicine.cmu.ac.th/dept/parasite/
nematode/wbmf.htm)
The Invaders . . .
Bacteria
Viruses
Fungi
Protista
Worms
...
Each year in sub-Saharan Africa, ∼12 million
people die……
animals too…..
Infectious diseases-HIV infection, malaria, and
t...
“Lack of effective point of care diagnostic
tests applicable in resource-poor endemic
areas is a critical barrier to effec...
Common Diagnostic Methods
Clinical signs
Animal inoculation
Smears
CMT
…….
Ab and Ag detection (ELISA…)
In vitro culture
D...
The Need…………..
WHO recommends that an ideal diagnostic test suitable for
developing, underdeveloped and undeveloped countr...
♞Polymerase chain reaction (PCR)
♞Ligase chain reaction (LCR)
♞Nucleic acid sequence-based
amplification (NASBA)
♞Strand D...
LAMP (Loop mediated isothermal amplification)
Originally reported by Notomi et al in 2000 of EIKEN Chemical
Co. Ltd., Japa...
Bst DNA polymerase with strand displacement
activity at 65℃
The whole amplification to 109 – 1010 copies is
finished withi...
The amplification efficiency is extremely high
Reduced total cost- not require special reagents or
sophisticated equipment...
Design of primers
4 types of primers based on the 6 distinct regions of
the target gene: the F3c, F2c and F1c regions at t...
FIP Forward Inner Primer (FIP) consists of the F2
region (at the 3' end) that is complementary to
the F2c region, and the ...
Loop Mediated Isothermal Amplification
Primer Regions
( Parida et al., 2008)
Two loop primers - forward loop primer (FLP) ...
Softwares…….
(www.premierbiosoft.com/isot
hermal/lamp.html)
(http://loopamp.eiken.co.jp/e/lamp/primer.html)
(Torres et al....
PRINCIPLE OF LAMP
(A) Non-Cyclic Steps : generation of stem loop
DNA with dumbbell-shaped structure at both
ends
One of the LAMP primers anneal to the complimentary
sequence of double stranded target DNA
Initiates DNA synthesis using t...
Through the activity of DNA polymerase with strand
displacement activity, a DNA strand complementary
to the template DNA i...
The F3 Primer anneals to the F3c region, outside of
FIP, on the target DNA and
Initiates strand displacement DNA synthesis...
A double strand is formed from the DNA strand
synthesized from the F3 Primer and the template
DNA strand
STEP 4
The FIP-linked complementary strand is released as
a single strand because of the displacement by the
DNA strand synthesiz...
This single strand DNA in Step (5) serves as a template
for BIP-initiated DNA synthesis and subsequent B3-
primed strand d...
Double stranded DNA is produced through the
processes described in Step (6)
STEP 7
The BIP-linked complementary strand displaced in
Step (6) forms a structure with stem-loops at each
end, which looks like ...
(A) Non-Cyclic Step : generation of stem loop
DNA with dumbbell-shaped structure at both
ends that is ready to enter into ...
(B) Cycling amplification step
Using Loop Primers
Time saving by Loop Primer……
Time required for amplification with Loop Primers is one-
third to one-half of that without L...
LAMP does not require thermal cycler
• Cycle reaction
• Denature (95℃)
• Annealing (50~60℃)
• Polymerization (72℃)
• ~ >1 ...
Detection
Visually
1.Fluorescence – Eye
2.Turbidity – Turbidmeter (or Eye)
Gel electrophoresis
LAMP product is visible to naked eyes
(DNA)n-1+dNTP → (DNA)n + P2O7
4-
P2O7
4- + 2Mg2+ → Mg2P2O7 ↓
PPT
{Biochemical and Bi...
Amplification products are stem-loop DNA structures with
several inverted repeats of the target and cauliflower-like
struc...
Turbidity increases - when concentration of
pyrophosphate ion exceeded 0.5 mM
on a 25-μl scale reaction, a DNA yield of mo...
Nature Protocols 3, 877 - 882 (2008)
Detection using a fluorescent metal indicator (calcein)
Green fluorescence
by free ca...
(a) Irradiating the tube using a handheld-UV lamp
(wavelength: 365 nm) from the bottom
(b) Under daylight. Plus sign denot...
(a) HNB: The color changes from violet
(negative reaction) to sky blue
(positive reaction)
(b) SYBR green and
(c) Calcein
...
Detection of 10-fold serially diluted λ DNA using
Hydroxy Naphthol Blue (HNB), Syber Green and Calcein
Visualization under...
Loopamp Realtime Turbidimeter (LA-500)
With the time and temperature preset, gene
amplification will occur, and the detect...
Analysis of the loop-mediated isothermal
amplification (LAMP) reaction products using
agarose gel electrophoresis (Tomita ...
(LaBarre et al.,2011)
(A) assembled incubator, (B) incubator lid with built-in spring timer, (C) CaO chamber
(w/ CaO added...
(Curtis et al.,2012)
Non-instrumented nucleic acid (NINA) heaters with DaqPRO 5300 Data recorder
Isothermal Amplification ...
LAMP Vs PCR
• LAMP does not require an expensive thermocycler
• Amplification specificity is extremely high as LAMP
requir...
LAMP can be done by rather crude DNA sample
PCR is susceptible to hemoglobin, Ig and Heparin
LAMP resists contamination of...
Practical applications
• As of April17, 2013, PubMed database has listed
more than 820 articles on this topic
• Before 2008, the Severe Acute Res...
LAMP - TB for the peripheral level
• LAMP reagent kit for detecting the M. tuberculosis
complex (Loopamp MTBC detection ki...
Collaborative development with FIND
(Foundation for Innovative New Diagnostics)
FIND: non-profit foundation for developing...
Overview of PURE Method
(Procedure for Ultra Rapid Extraction)
( Okamoto, 2011)
PURE-TB LAMP Operation
( Okamoto, 2011)
LAMP Reagents in Dried Form
Dried LAMP reagents fixed
on the lids
↓
Reconstitute by PURE
treated sample solutions
Save ste...
• Malaria LAMP kit (Loopamp MALARIA Pan/Pf
detection kit) has been launched (diagnostic
accuracy of the kit is similar to ...
Thailand: Cholera outbreaks in Tak province, 2008
(Okamoto, 2011)
• Application of LAMP to the world’s three major
diseases - TB, malaria, and human
immunodeficiency virus (HIV), has also ...
Magnetic bead-based assay for MRSA rapid detection –
what an attractive idea!
(Wang et al., 2011, Lab Chip )
A proposed three-step LAMP
method for diagnosis of
neglected tropical diseases
(Njiru, 2012)
MALE FEMALE
The natural light observation of the reaction tube
using digital camera: Colour change after addition
of EtBr ...
MALE FEMALE
The natural light observation of the reaction tube
using digital camera: Precipitate formation after
addition ...
In India………..
LAMP is highly sensitive and specific DNA/RNA amplification
method
Advantage of LAMP is isothermal reaction condition, her...
“Innovations in biotechnology that combine molecular
biology, microfabrication and bioinformatics are
moving nucleic acid ...
THANK
YOU
LAMP (Loop Mediated Isothermal Amplification)
LAMP (Loop Mediated Isothermal Amplification)
LAMP (Loop Mediated Isothermal Amplification)
LAMP (Loop Mediated Isothermal Amplification)
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LAMP (Loop Mediated Isothermal Amplification)

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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.

Published in: Education, Technology

LAMP (Loop Mediated Isothermal Amplification)

  1. 1. (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”
  2. 2. (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)
  3. 3. 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)
  4. 4. “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)
  5. 5. 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
  6. 6. 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)
  7. 7. ♞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)
  8. 8. 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
  9. 9. 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
  10. 10. 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)
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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)
  15. 15. PRINCIPLE OF LAMP (A) Non-Cyclic Steps : generation of stem loop DNA with dumbbell-shaped structure at both ends
  16. 16. 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
  17. 17. 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
  18. 18. 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
  19. 19. A double strand is formed from the DNA strand synthesized from the F3 Primer and the template DNA strand STEP 4
  20. 20. 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
  21. 21. 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
  22. 22. Double stranded DNA is produced through the processes described in Step (6) STEP 7
  23. 23. 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
  24. 24. (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
  25. 25. (B) Cycling amplification step
  26. 26. Using Loop Primers
  27. 27. 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
  28. 28. 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
  29. 29. Detection Visually 1.Fluorescence – Eye 2.Turbidity – Turbidmeter (or Eye) Gel electrophoresis
  30. 30. 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
  31. 31. 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)
  32. 32. 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
  33. 33. Nature Protocols 3, 877 - 882 (2008) Detection using a fluorescent metal indicator (calcein) Green fluorescence by free calcein
  34. 34. (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)
  35. 35. (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)
  36. 36. 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)
  37. 37. 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
  38. 38. Analysis of the loop-mediated isothermal amplification (LAMP) reaction products using agarose gel electrophoresis (Tomita et al., 2008)
  39. 39. (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
  40. 40. (Curtis et al.,2012) Non-instrumented nucleic acid (NINA) heaters with DaqPRO 5300 Data recorder Isothermal Amplification Using a Chemical Heating Device
  41. 41. 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)
  42. 42. 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
  43. 43. Practical applications
  44. 44. • 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)
  45. 45. 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
  46. 46. Collaborative development with FIND (Foundation for Innovative New Diagnostics) FIND: non-profit foundation for developing diagnostic tools for poverty-related diseases
  47. 47. Overview of PURE Method (Procedure for Ultra Rapid Extraction) ( Okamoto, 2011)
  48. 48. PURE-TB LAMP Operation ( Okamoto, 2011)
  49. 49. 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)
  50. 50. • 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
  51. 51. Thailand: Cholera outbreaks in Tak province, 2008 (Okamoto, 2011)
  52. 52. • 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
  53. 53. Magnetic bead-based assay for MRSA rapid detection – what an attractive idea! (Wang et al., 2011, Lab Chip )
  54. 54. A proposed three-step LAMP method for diagnosis of neglected tropical diseases (Njiru, 2012)
  55. 55. 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)
  56. 56. 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)
  57. 57. In India………..
  58. 58. 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
  59. 59. “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.”
  60. 60. THANK YOU

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