Towards development of easy-to-use molecular diagnostic method for HAT ~ Loop-mediated isothermal amplification of DNA ~ O. M. M. Thekisoe 1 , M. Inoue 1 , J. Ndung’u 2 , N. Inoue 1 * 1 OIE Reference Laboratory on Surra National Research Center for Protozoan Diseases Obihiro University of Agriculture and Veterinary Medicine, 2 Head of HAT Diagnostics Programme, Foundation for Innovative New Diagnostics (FIND), *Corresponding author: N. Inoue, E-mail ircpmi@obihiro.ac.jp
Common Diagnostic Methods for Trypanosomosis Clinical signs Field Methods Easy-to-Use Giemsa smears Affordable Wet blood film Unspecific clinical signs HCT/BCT Low specificity Low sensitivity Animal inoculation In vitro culture Ab and Ag detection (ELISA/IFAT) High specificity Xenodiagnosis High sensitivity DNA hybridization Complicated Expensive PCR Time consuming Laboratory Methods
Control Strategies for HAT Vaccine Not available Treatment Available but high risk Need accurate diagnosis at early phase Diagnosis Available but need improvement Tsetse control Fairly good but imperfect
Control Strategies for HAT Vaccine Vaccine development ? Treatment New drug ? Diagnosis New diagnostics Tsetse control Eradication ? Which is the best HAT control strategy?
To Control HAT Need Accurate Sensitive Affordable Easy-to-use Molecular Diagnostic tests
Our Answer Loop-mediated isothermal gene amplification 1. A single step isothermal amplification 2. Uses 4 primers: Highly sensitive and specific 3. Rapid: Within 60 min. 4. Visual detection of the results 5. No sophisticated equipment required
Loop-mediated isothermal gene amplification (LAMP) In 2000, Notomi, T. et al. reported a new method, termed LAMP, that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. The LAMP employs a Bst DNA polymerase and a set of 4 specially designed primers that recognize 6 distinct sequences on the target DNA. Except for the primer set, all reagents for LAMP is available as a kit, called “Loopamp DNA amplification kit” (Eiken Chemical Co. Ltd, Japan).
LAMP does not require thermal cycle. Reaction mix preparation PCR LAMP Cycle reaction Isothermal reaction Denature (95 ℃ ) 62~65 ℃ Annealing (50~60 ℃ ) Max 1 hour Polymerization (72 ℃ ) ~3 hours Detection
Detection Gel electrophoresis Visually 1.Fluorescence – Eye 2.Turbidity – Turbidmeter (or Eye)
Visual Detection by Turbidity Precipitating reaction (Mori, Y. et al., 2001) (DNA) n-1 + dNTP → (DNA) n + P 2 O 7 4- → Mg 2 P 2 O 7 4- + 2Mg 2+ P 2 O 7 Negative Positive Sometimes, it is difficult to distinguish positive from negative. Need turbidmeter for accurate judgment.
Visual Detection by Fluorescence Reagent Calcein Loopamp Fluorescent Detection Reagent Only by mixing this reagent with Loopamp DNA Amplification Kit or Loopamp RNA Amplification Kit (RT-LAMP) before the reaction, results can be visually observed. About instructions for using this reagent, refer to the package insert. UV irradiation requirements for Loopamp Fluorescent Detection Reagent.
Results Under UV light Positive Negative
Results Under Sun light Positive Negative The same experiment was done at my room yesterday.
The Selected Target Genes Sub-genus and sub-species markers Trypanozoon PFRA ~28 copies T. b. gambiense TgsGP Single copy T. b. rhodesiense SRA Single copy
Sensitivity of HAT-LAMP Sensitivity LAMP Primer Practical Analytical PFRA1-LP174 100 fg 100 copies (1 cell) TgsGP05-LP21 1 pg Ongoing (10 cells) SRA06-LP01 1 pg Ongoing (10 cells)
Test sample for LAMP LAMP requires template DNA as a test sample. Clinical samples: BLOOD, SPUTUM, FECES ⇒ DNA/RNA extraction DNA = Directly use as template DNA (DNA virus, Bacteria, Protozoa) RNA = Reverse transcription required before LAMP reaction (RNA virus) Towards field diagnosis: DNA/RNA extraction and reverse transcription have to be designed as simple as possible.
Simple DNA extraction method Use of Filter Paper (FTA card) BLOOD, SPUTUM, FECES ⇒ DNA extraction To simplify DNA extraction procedures, we used commercially available reagents, the FTA card and FTA reagent (Whatman). FTA card is a chemically treated filter paper that allows for the rapid isolation of pure DNA. Other filter-based simple DNA extraction procedures also available.
Simplification of Sample Preparation Procedures Different LAMP template preparation 1. Fresh infected blood: Apply 1 µ l infected blood to LAMP. 2.Hemolysed blood: 10 µ l of infected blood was hemolysed with 1 ml distilled water, and spun at 500 xg for 10 min. Ppt was suspended in 10 µ l TE. Use 1 µ l for LAMP 3.FTA card 4.Genomic DNA (Phenol-chloroform method)
LAMP Reactions on Different DNA Preparation Results 100% 75 - 80% 67 - 72% LAMP Reagent Storage 48 - 55% Temperatures * **** ** *** *Crude blood; **Hemolysed blood; ***Filter paper; ****genomic DNA
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.
LAMP Demonstration at LIRI, Tororo, Uganda
Result CSF sample from a HAT patient T. congolense (-) T. brucei group (+) T. b. gambiense (-)
Conclusions and Perspective 1. 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 thermalcycler. 2. Although recommended reagent storage temperature is -20 o C, reagents can be stored at ambient temperature for at least 2 weeks. Hereby there is no need to have cold chain for reagent distribution. 3. Crude DNA preparation can be used as LAMP template DNA. 4. Cost of LAMP can be reduced to approximately 1 USD/test or cheaper. (EIKEN co. ltd. scientists, personal communication). 5. We need to develop “All-in-one LAMP diagnosis kit”, which includes sample collection tools, easy DNA extraction and LAMP reagents. 6. LAMP can be a field molecular diagnostic method for infectious diseases.
Acknowledgements Dr. Oriel Thekisoe, NRCPD, Obihiro Univ., Japan – Most of this presentation is his Ph.D. thesis Dr. Chihiro Sugimoto, CZC, Hokkaido Univ., Japan Dr. Tugunori Notomi, EIKEN Chemical Co. Ltd., Japan Dr. Charles Otim, LIRI, Uganda Dr. Joseph Magona, LIRI, Uganda Dr. John Omolo, CVL, Tanzania Dr. Heriel Mbwambo, CVL, Tanzania Dr. Joseph Ndung’u, FIND, Switzerland
Thank you!
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