Towards development of easy-to-use molecular diagnostic method for - - PowerPoint PPT Presentation

Towards development of easy-to-use molecular diagnostic method for HAT

~ Loop-mediated isothermal amplification of DNA ~

• O. M. M. Thekisoe1, M. Inoue1, J. Ndung’u2, N. Inoue1*

1OIE Reference Laboratory on Surra

National Research Center for Protozoan Diseases Obihiro University of Agriculture and Veterinary Medicine,

2Head 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

High specificity High sensitivity Complicated Expensive Time consuming Animal inoculation Giemsa smears HCT/BCT Wet blood film Clinical signs Field Methods Easy-to-Use Affordable Unspecific clinical signs Low specificity Low sensitivity PCR DNA hybridization Xenodiagnosis Ab and Ag detection (ELISA/IFAT) In vitro culture Laboratory Methods

Control Strategies for HAT

Vaccine Not available Treatment Available but high risk Diagnosis Available but need improvement Tsetse control Fairly good but imperfect

Need accurate diagnosis at early phase

Control Strategies for HAT

Vaccine Vaccine development ? Treatment New drug ? Diagnosis New diagnostics Tsetse control Eradication ? Which is the best HAT control strategy?

Need Accurate Sensitive Affordable Easy-to-use

Molecular Diagnostic tests

To Control HAT

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.

Except for the primer set, all reagents for LAMP is available as a kit, called “Loopamp DNA amplification kit” (Eiken Chemical Co. Ltd, Japan).

The LAMP employs a Bst DNA polymerase and a set

• f 4 specially designed primers that recognize 6

distinct sequences on the target DNA.

LAMP does not require thermal cycle.

Reaction mix preparation

Detection

Cycle reaction Denature (95℃) Annealing (50~60℃) Polymerization (72℃) ~3 hours Isothermal reaction 62~65℃ Max 1 hour PCR LAMP

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

Visual Detection by Turbidity

Negative Positive → Mg2P2O7 (DNA)n-1+ dNTP → (DNA)n + P2O7

4-

P2O7

4- + 2Mg2+

Precipitating reaction (Mori, Y. et al., 2001) 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.

Positive Negative

Results Under UV light

The same experiment was done at my room yesterday.

Positive Negative

Results Under Sun light

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

Practical Analytical PFRA1-LP174 100 fg 100 copies (1 cell) TgsGP05-LP21 1 pg Ongoing (10 cells) SRA06-LP01 1 pg Ongoing (10 cells) Sensitivity LAMP Primer

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. Test sample for LAMP

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.

Simple DNA extraction method

Use of Filter Paper (FTA card)

Simplification of Sample Preparation Procedures

Different LAMP template preparation

• 1. Fresh infected blood: Apply 1 µl infected blood to LAMP.

2.Hemolysed blood: 10 µl of infected blood was hemolysed with 1 ml distilled water, and spun at 500 xg for 10 min. Ppt was suspended in 10 µl TE. Use 1 µl for LAMP 3.FTA card 4.Genomic DNA (Phenol-chloroform method)

LAMP Reactions on Different DNA Preparation

*Crude blood; **Hemolysed blood; ***Filter paper; ****genomic DNA

*** ** **** *

75 - 80% 67 - 72% 48 - 55%

100%

Results

LAMP Reagent Storage Temperatures

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

• T. congolense (-)
• T. brucei group (+)
• T. b. gambiense (-)

Result CSF sample from a HAT patient

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 -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.

• 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!