Loop-mediated Isothermal gene Amplification (LAMP)clione.ne.jp/~sample/sentan/jsps/img/Oriel-Kenya.pdfLoop-mediated Isothermal gene Amplification (LAMP): Towards Field Molecular Diagnosis

Loop-mediated Isothermal gene Amplification (LAMP):

Towards Field Molecular Diagnosis of African Trypanosomosis

Oriel M. M. Thekisoe

National Research Center for Protozoan DiseasesObihiro University of Agriculture and Veterinary Medicine

Obihiro, Hokkaido, Japan

Common Diagnostic Methods for Trypanosomosis

Gene Amplification• PCR

Serology• ELISA• IFAT• CATT

Microscopy• Thin & thick blood smears• Wet smears• Buffy coat

Why LAMP for Diagnosis of Trypanosomosis?

Loop-mediated Isothermal Amplification

LAMP simple, specific, sensitive, rapid & cost effective diagnostic gene amplification method

(Notomi et al., 2000)

Common diagnostic methods for trypanosomosis are limited by lack

of specificity, sensitivity, whilst some are complicated and

expensive

LAMP Reagents, Amplification and Detection

Detection

Turbidity

ー ＋

Fluorescent DyeGel Electrophoresis

Reagents

Buffer + dNTPs 12.5 µl

Primer mix 0.9 µl(FIP, BIP, F3 & B3)

DNA 2.0 µl

Bst DNA Poly. 1.0 µl

DDW (H20) 8.6 µl

Amplification

63~65℃Maximum of 1 hour

25 ul

＋ ー

Loop-mediated Isothermal Amplification for Detection of African Trypanosomes

N. Kuboki, N. Inoue, T. Sakurai, F. Di Cello, D. J. Grab, H. Suzuki, C. Sugimoto, I. Igarashi

J. Clin. Microbiol. 41:5517 - 5524 (2003)

Objectives

To develop a specific LAMP for diagnosis of T. bruceigroup and T. congolese

LAMP primers for Trypanosoma congolense andT. brucei genes

Target gene: P0 (T. congolense ribosomal P0 subunit)Primer set: P01

F3 : 5’-CGTGGTAAGGGTGAATTGGT-3’B3 : 5’-GTGTCCGTCCAACACCTTCFIP : 5’-ATCCGTCGCCTTGCTGTCC TTTTT ATGGGGAAGAAGACGCTTCA-3’BIP : 5’-CAAGCAGCTGCTGTGCGGTA TTTTT GATCTCCGTAACGTCCTCG-3’

Target gene: PFR A (T. brucei paraflagellar rod protein A)Primer set: A1F3 : 5’-TCACAACAAGACTCGCACG-3’B3 : 5’-GGGCTTTGATCTGCTCCTC-3’FIP : 5’-TCAGAAGCGTCGAGCTGGGA TTTT ATCGACAATGCCATCGCC-3’BIP : 5’-CGCAAGTTCCTGTGGCTGCA TTTTTT CCCAAGAAGAGCCGTCT-3’

Specificity of LAMP Primers for T. brucei and T. congolense

M N P B C B C B C B CControl A1 A2 P01 P02

M: 1 kbp ladder, N: Negative control, P: Positive controlB: T. brucei, C: T. congolense

Template DNA (pg)

PCR

(A) LAMP and PCR

10 kbp

1 kbp

104

103

102 110

A1

M 1 2 3 4 5

104

103

102 110

P01

M 6 7 8 9 10

10 kbp

1 kbp

A1

1 2 3 4 5

P01

6 7 8 9 10

(B) Southern blotting

Sensitivity (A) and specificity (B) of LAMP

M 1 2 3 4 5 6 7 8 9 10 11 12

1 kbp

5 kbp10 kbp

Specificity of PFR A LAMP

T. b. g

ambie

nse IL2

343

T. b. rh

odes

iense

IL150

1

T. b. g

ambie

nse IL3

253

T. eva

nsi

Toxop

lasma g

ondii

Babes

ia eq

ui

Babes

ia ca

balli

Neosp

ora ca

ninum

Vero (M

onke

y)

HCT-8 (H

uman

)

MDBK (Bov

ine)

NIH/3T

3 (Mou

se)

In actual field diagnoses of trypanosomoses, whole blood samples are the most common test material for the examination.

FTA card is a chemically treated filter paper that allows for the rapid isolation of pure DNA.

To simplify DNA extraction procedures, we used commercially available reagents, the FTA card and FTA reagent (Whatman).

Therefore, mice were injected with T. b. gambiense IL3253, which has a low virulence in mice. Every other day blood samples were collected from the tail vein.

Further evaluation of PFR A LAMP

FTA cardDNA extraction From FTA Card

Punch samples

Wash with FTA reagentPrepared samples

LAMP Modified PCRAmpdirect-D (shimazu Biotech Co., Japan), which enhanses PCR amplification, were contained in reaction mixture.

4 12 142 6 8 10 16 18 20 22 24 26 28 30M

+ + + +

5 kbp10 kbp

1 kbp

PCR

Buffy coat

DPI 0

Sequential analysis of blood samples from mouse (# 1) infectedwith T. brucei gambiense (IL 3253)

Mouse 2

Mouse 5

Mouse 4

Mouse 3

LAMP + + + + + + + + + - + + + + +

LAMP + + + + + + + + + + + + + + +

LAMP + + + + + + + + + - + + + + +

LAMP + + + + + + + + + - + + + + +

PCR - + - - - + - - - - - - - - -

PCR - - - - - - - - - - - - - - -

PCR - - - - - - - - - - - - - - -

PCR - - - - - - - - - - - - - - -

Microscopy - + - - - + - - - - - - - - -

Microscopy - - - - - - - - - - - - - - -

Microscopy - - - - - - - - - - - - - - -

Microscopy - + - - - - + - - + + - - - +

Days post-infection2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Detection of T. b. gambiense Infection in Experimentally Infected Mice

Conclusions

• T. brucei (PFR A) and T. congolense (P0) genes are specifically and rapidly amplified by LAMP.

• LAMP reaction is specific for the T. brucei group and can detect a T. brucei gambiense infection in mice with greater sensitivity than microscopic tests or PCR.

• However, PFRA LAMP primers also amplify T. evansiDNA, because the gene is conserved amongst species and sub-species of Trypanozoon.

Evaluation of LAMP, PCR and parasitological tests for detection of T. evansi in experimentally

infected pigs

O. M. M. Thekisoe, N. Inoue, N. Kuboki, D. Tuntasuvan, W. Bunnoy, S. Borisutsuwan, I. Igarashi, C. Sugimoto.

Vet. Parasitol. 130: 327- 330 (2005)

Objectives

• To determine efficiency of LAMP as a diagnostic method for T. evansi infections.

• To conduct comparative evaluation of LAMP, PCR and parasitological methods for detection of T. evansiinfections.

Background

• Trypanosoma evansi causes epidemics of disease commonly called surra and is mechanically transmitted by the tabanid flies.

• It is distributed in Asia, Africa & South America and it infects buffaloes, cattle, horses, donkeys, camels, goats, sheep and pigs.

• Microscopic and serological diagnostic methods are limited by lack of sensitivity, specificity and by the presence of cross reactivity.

• PCR has been reported as sensitive and specific but is not commonly used in surra endemic countries as it requires an expensive automated thermal cycler.

• Recent reports of T. evansi infection in humans in India (WHO, 2005), highlighted the significance of accurate diagnosis of this protozoan parasite.

Materials and Methods

6 pigs were infected with T. evansi & 2

were used as negative controls

Blood CollectionWeekly for a period of

12 weeks

DNA Amplification

LAMP&

PCR

Mouse inoculationMicrohematocrit centrifugeThin Blood smear

Diagnostic Methods

LAMP & PCR Primers

LAMP target gene: PFR A, Primer set A1 (Kuboki et al., 2003) F3 : 5’- TCACAACAAGACTCGCACG -3’B3 : 5’- GGGCTTTGATCTGCTCCTC -3’FIP : 5’-TCAGAAGCGTCGAGCTGGGATTTTATCGACAATGCCATCGCC-3’BIP : 5’- CGCAAGTTCCTGTGGCTGCATTTTTT CCCAAGAAGAGCCGTCT -3’

PCR target gene: T. evansi minicircle DNA primers (Artama et al., 1991)MP1: 5’-CAACGACAAAGAGTCAGT-3’MP2: 5’-ACGTGTTTTGTGTATGGT-3’

Results

Detection sensitivity and specificity of diagnostic methods

Method No gold standard (%) MI as gold standard (%)

Sensitivity Specificity Sensitivity Specificity

MI** 65 100 nd* ndLAMP 45 100 49 61PCR 33 100 44 87MHCT*** 38 100 53 91TBS**** 24 100 35 96

*nd – not done**MI- mouse inoculation***MHCT- Microhematocrit test****TBS- Thin blood smear test

Chi-square test for calculation of significant differences (p<0.05)

No Gold standard MI as gold standard

Method Chi-square P-value Chi-square P-valueMI vs LAMP 5.2 0.0229 nd ndMI vs PCR 13.3 0.0003 nd ndMI vs MHCT 9.8 0.0017 nd ndMI vs TBS 22.3 0.0001 nd ndLAMP vs PCR 2.0 >0.05 0.9 >0.05LAMP vs MHCT 0.8 >0.05 0.5 >0.05LAMP vs TBS 6.5 0.0105 2.7 >0.05PCR vs MHCT 0.3 >0.05 0.1 >0.05PCR vs TBS 1.3 >0.05 0.5 >0.05MHCT vs TBS 2.9 >0.05 1.1 >0.05

Conclusion

• MI (mouse inoculation test) was the most sensitive method but is not practically applicable for diagnosis as its time consuming.

• LAMP showed relatively higher detection sensitivity than PCR as similarly reported by Kuboki et al. (2003).

• This study validates LAMP as an alternative gene amplification method for the diagnosis T. evansi infections.

Oriel M.M. Thekisoe, Andrew Nambota, Jun Yasuda, Noritaka Kuboki, Kozo Fujisaki, Ikuo Igarashi, Chihiro Sugimoto, Noboru Inoue

Manuscript Submitted - under review

Species-Specific Loop-Mediated Isothermal Amplification (LAMP) for Diagnosis of Trypanosomiasis and Its Application

to Epidemiological Studies

Objectives of the Study

• Specific LAMP for detection of T. b. gambiense, T. b. rhodesiense, T. evansi, T. congolense, and T. cruzi.

• Application of LAMP in epidemiological studies

LAMP Primers and Target GenesT. b. gambiense: 5.8S -ITS1F3 : 5’-AAGCTCTCTCGAGCCATC-3’B3 : 5’-TGACATACACAATATGTGCGA-3’FIP : 5’-GCGTTGAACAACACAAAATAGGTGATGCCACATTTCTCAGTGT-3’BIP : 5’-CCACCTCTTCTCCTCGTGTGGAAGAAAGAGATGAAAGATATCGTA-3’

T. b. rhodesiense: SRAF3 : 5’-TCACAACAAGACTCGCACG-3’B3 : 5’-GGGCTTTGATCTGCTCCTC-3’FIP : 5’-TCAGAAGCGTCGAGCTGGGATTTTATCGACAATGCCATCGCC-3’BIP : 5’-CGCAAGTTCCTGTGGCTGCATTTTTTCCCAAGAAGAGCCGTCT-3’

T. evansi: RoTat 1.2 VSGF3 : 5’-GCCGCCAATGTAGCTCTT-3’B3 : 5’-CCGCTGCTCGTATGTGTC-3’FIP : 5’-TTCGATCGCTGCGAAGTGCGTCTGGAAGCCATTGTGCG-3’BIP : 5’-AAGCTCTTGATTTACGCGGCGGGCTGCTAACCCTCTTGCTG-3’

T. congolense: 18S rRNAF3 : 5’-TGTGTGTTTGTCGTGGAAGC-3’B3 : 5’-ATTCGTGACCGCGTCAAA-3’FIP : 5’-GCGCATGCGTCGGTGTTATTTTCGCGTGTGTGTTCATGTCA -3’BIP : 5’-ACTCTCCCCCCAAAATGGTTGTCCAAGCACGCAAATTCACAT -3’

T. cruzi: 18S rRNAF3 : 5’-GGACGTCCAGCGAATGAATG-3’B3 : 5’-CCTCCGTAGAAGTGGTAGCT-3’FIP : 5’-GGTAAAAAACCCGGCTTTCGCAACCGGCAGTAACACTCAGA-3’BIP : 5’-CGATGGCCGTGTAGTGGACTGTTTCTCAGGCTCCCTCTCC-3’

Specificity of LAMP vs PCRT. b. gambiense T. congolense

T. cruzi T. evansi

Results

T. b. g

ambie

nse

M T. b.br

ucei

T. b. r

hode

siens

eT. b

. con

golen

se

T. eva

nsi

T. cru

ziT. p

arva

B. bov

isT. g

ondii

N. can

inum

M T. con

golen

seT. b

.bruc

eiT. b

.gambie

nse

T. eva

nsi

T. cru

ziT..b

. rho

desie

nse

T. par

vaB. b

ovis

B. bige

minaN. c

aninu

m

M T. cru

ziT.b.

bruc

eiT. e

vans

iT. b

. rho

desie

nse

T. con

golen

seT. p

arva

B. bov

isB. e

qui

T. gon

dii

M T. eva

nsi

T. b. b

ruce

iT. c

ongo

lense

T. rho

desie

nse

T. par

vaT. o

rient

alis

B. bov

isB. b

igemina

B. equ

iB. c

aball

i

LAMP LAMP

PCR

LAMP LAMP

PCR

PCR PCR

M 100ng 10ng 1ng 100pg 10pg 1pg 100fg 10fg 1fg

PCR

Sensitivity of LAMP vs PCR

M 100ng 10ng 1ng 100pg 10pg 1pg 100fg 10fg 1fg

PCR

T. b. gambiense

PCR

M 100ng 10ng 1ng 100pg 10pg 1pg 100fg 10fg 1fgT. congolense

M 100ng 10ng 1ng 100pg 10pg 1pg 100fg 10fg 1fg

PCR

T. cruzi T. evansi

LAMP LAMP

LAMP LAMP

Trypanosome Country Animal spp Total # positive # % positive

T. congolense Zambia Cattle 40 5 12.5

T. congolense Zambia Wildebeest 20 2 10

T. congolense Zambia Zebra 8 0 0

T. congolense Zambia Impala 8 0 0

T. congolense Zambia Buffalo 8 0 0

T. evansi Brazil Cattle 200 5 2.5

T. evansi Brazil Horse 22 0 0

T. evansi Mongolia Cattle 240 1 0.4

Application of LAMP in Epidemiological Studies

RED: DNA from FTA cardBLACK: DNA from total blood

All samples were PCR negative

• Species specific LAMP has been successfully developed for T. b. gambiense, T. evansi, T. congolense and T. cruzi.

• LAMP has been applied in epidemiological studies for T. congolense and T. evansi.

• Development of LAMP for T. b. rhodesiense was unsuccessful as there were no reactions occurring with primers designed from SRA gene.

Conclusions

Stability of LAMP Reagents Stored at Different temperatures, itsAmplification Efficiency on Different DNA Templates and its

Tolerance to Inhibitory Substances

Oriel M.M. Thekisoe, Raoul B. S. Bazie, Andrea Coronel-Servian, Chihiro Sugimoto, Kozo Fujisaki, Noboru Inoue

Manuscript Submitted – under review

Background

• Due to its simplicity, LAMP is a strong candidate for application of molecular diagnosis in the field where cost and environmentalconstraints prohibit the use of PCR.

• However, conditions in the field differ significantly to laboratory conditions.

• For example, temperature of the surroundings, availability of equipment for reagent storage and DNA extraction.

• There is a need to conduct experiments under conditions similar to field, clinics or hospitals, with simplified DNA template preparation methods.

Objectives

• To evaluate stability of LAMP reagents when stored at 25ºC and 37ºC.

• To assess detection efficiency of LAMP on different DNA templatepreparations.

• To determine the tolerence of LAMP on PCR inhibitory substances.

Materials & Methods1st Set of Experiments

LAMP Reagents (LAMP buffer with dNTP’s, Primers, DDW & Bst DNA Polymerase)were kept in 25ºC and 37ºC incubators.

LAMP was conducted for amplification of T. brucei brucei and T. congolense DNAFor a period of 15 days of reagent storage.

2nd Set of Experiments

5 Balb/c were infected with T. congolense (IL1180) & 5 were kept as controls.LAMP with reagents kept in 25ºC and 37ºC incubators for 30 days of reagent storage.

Blood was collected from mice and different DNA templates were prepared:(i) Crude blood

(ii) Hemolysed blood(iii) Filter paper

(iv) Genomic DNA (gDNA: phenol-chloroform extraction)Parasitaemia was also monitored for the entire period of experiment.

LAMP with reagents stored at -20ºC (Recommended storage temperature) was also conducted as standard method

3rd set of experiments

PCR Inhibitory substances

• EDTA• Hemoglobin from bovine blood• Heparin• IgG• IgM

Were prepared in concentrations of 1 mg/ml, then diluted serially and added into the LAMP and PCR reaction.

Day0 Day 1 Day 3 Day 6 Day 9 Day 12 Day 15

M Tb N Tb N Tb N Tb N Tb N Tb N Tb N

Day0 Day1 Day3 Day6 Day 9 Day12 Day15

M Tb N Tb N Tb N Tb N Tb N Tb N Tb N

25ºC 37ºC

Amplification of T. brucei brucei genomic DNA by LAMP

Tb – T. brucei bruceiN – Negative control (T. congolense DNA)

Parasitaemia of mice infected with T. congolense (IL 1180)

0

50

100

150

200

250

300

350

400

450

500

0 4 8 12 16 20 24 28 30

Days post-infection

Para

siat

emia

( x1

06 )

mouse 1mouse 2mouse 3mouse 4mouse 5

01020304050

60708090

100

Crude Hemo Filt. Paper gDNADNA template

Posi

tive

LAM

P re

actio

n (%

)

-20 оC25 оC37 оC

Detection Performance of LAMP on Different DNA Templates

Crude: Crude blood Filt. Paper: FTA card methodHemo: Hemolysed blood gDNA: purified genomic DNA (PCI method)

Effects of DNA Polymerase Inhibitors on LAMP & PCR

PCR LAMP

1.0 0.1 0.01 0.001 0 1.0 0.1 0.01 0.001 0Heparin - - + + + - + + + +Heme - - - + + + + + + +EDTA - - + + + - + + + +IgG - - + + + + + + + +IgM - + + + + + + + + +

(mg/ml) (mg/ml)

Effects of DNA Polymerase Inhibitors on LAMP & PCR

PCR LAMP

1.0 0.1 0.01 0.001 0 1.0 0.1 0.01 0.001 0Heparin - - + + + - + + + +Heme - - - + + + + + + +EDTA - - + + + - + + + +IgG - - + + + + + + + +IgM - + + + + + + + + +

(mg/ml) (mg/ml)

Conclusions

• LAMP with reagents stored at 25ºC and 37ºC can amplify trypanosome DNA which are possible ambient temperatures in the field.

• No significant difference of detection sensitivity between LAMP reagents stored at -20ºC, 25ºC and 37ºC.

• Best LAMP performance was achieved with gDNA template (100 %), followed by filter paper (78 %), crude (72 %) and hemolysed blood (51 %).

• LAMP reactions were only inhibited by high concentrations (1 mg/ml) of EDTA and Heparin.

• Further improvement of simple DNA extraction method would enhance chances of application of LAMP in the field.

Development of LAMP for Detection of Bovine Theileriosis

1. Clinical signs

2. Microscopic examination-Blood smear

3. Immunodiagnosis-Complement fixation test (CFT) -Indirect fluorescent antibody test (IFAT)-Enzyme-linked immunosorbent assay (ELISA)-Immunochromatographic test (ICT)

4. Genetic diagnosis-Polymerase chain reaction (PCR)

Diagnosis of piroplasmosisDiagnosis of piroplasmosis

IFAT

PCR

MICROSCOPY

Objective

To develop LAMP for specific detection of Theileria parva

Target genes

p104

p67

HSP 70

18S rRNA

LAMP primers for the amplification of LAMP primers for the amplification of T. parvaT. parva

Target geneTarget gene SequenceSequence

HSPHSP7070FIP:5FIP:5’’--TGGGTTACGGGCTTCTTGGTTTCCTACGTCGCATTCACTGACTGGGTTACGGGCTTCTTGGTTTCCTACGTCGCATTCACTGAC--33’’BIP:5BIP:5’’--ATTTTCGACGCCAAGAGGCTCAAATGGCCAGTGCTTCATGTCATTTTCGACGCCAAGAGGCTCAAATGGCCAGTGCTTCATGTC--33’’F3:5F3:5’’--GGAAACAGGACAACGCCGGGAAACAGGACAACGCCG--33’’F3:5F3:5’’--CCGTTTGGTCCGTTGGTAACCGTTTGGTCCGTTGGTAA--33’’

p104p104TATGACTATGACGATTACAGTAAAGCTGCAAGATTACAGTAAAGCTGCAAGTAGGTAGGTCTAAGGAAACTAAGACAACGTCTAAGGAAACTAAGACAACAGTTAGTTGCATTTGCATTT

GATGAATCATTTGGATGAATCATTTGAATCAAATCAATACTTAAGGAAACTCTCCCTGAGAATACTTAAGGAAACTCTCCCTGAGACACCACCTGGAACTCCCTTTACTGGAACTCCCTTTA

CCACTCCCCACTCCTAGACCTTAGACCTGTGCCACCAAAACGTCCCAGTGCCACCAAAACGTCCCAGGACACCTGAAAGTCCGGACACCTGAAAGTCCCTTTGAGCCACCTTTGAGCCAC

CTAAGGACTAAGGATCTCCTGATTCACCGTCTAGTAGCTGATTCACCGTCTAGTAGTCCATCTGAGTTTTTTACACCGCCAGAAAGCTCCATCTGAGTTTTTTACACCGCCAGAAAGC

B3

F3 F2 Loop F

F1c

B2

B1c

Loop B

F3

FIPBIP

B3

LoopF & LoopB

T. parva Lamp for p104 geneSpecificity Test

M

T. par

vaT. a

nnulata

T. mutan

sT. ta

urotra

giT. o

riental

isB. b

ovis

T. bru

ceiBov

ine bloo

d

T. parva Lamp for p104 geneSensitivity

M 100ng 10ng 1ng 100pg 10pg 1pg 100fg 10fg 1fg DDW

PCR 181bp

LAMP Primers targeting HSP70 gene of Theileria parva

LAMP

Specificity

M 100ng 10ng 1ng 100pg 10pg 1pg 100fg 10fg 1fg

LAMP

PCR 181bp

Sensitivity

M

T. parv

aT. a

nnulata

T. mutan

sT. ta

urotra

giT. o

riental

isB. b

ovisT. b

ruce

iBovin

e blood

Method Arusha Dar es Salaam

Theileria sp +* (%)

T. congolense+* (%)

Theileria sp +* (%)

T. congolense+* (%)

LAMP 0/24 (0 %) 4/24 (16. 6 %) 18/40 (45 %) 12/40 (30 %)

PCR 0/24 (0 %) 1/24 (4.1 %) 9/40 (22.5 %)

0/40 (0 %)

TBS** 0/24 (0 %) 0/24 (0 %) 0/40 (0%) 0/40 (0%)

Detection performance of LAMP, PCR and Blood SmearsHSP70 Primers

+* = Number of positively detected samples** = Thin blood smear

Conclusions

• HSP70 primers can be used as universal primers for bovine theileriosis.

• HSP70 primers – Higher sensitivity than PCR & microscopy.

• P104 primers specifically detect T. parva, however sensitivity needs improvement.

• p67 primers – No reaction

• 18s rRNA primers are unspecific.

General Summary of LAMP target genes

1.T. brucei brucei PFRA Universal2.T. brucei rhodesiense PFRA Universal

SRA Ongoing3.T. brucei gambiense PFRA Universal

5.8S-ITS2 Specific4.T. congolense ribosomal P0 Specific

18S rRNA Specific

Parasite Genes Specificity

T. parva HSP70 & 18s rRNA Universalp104 Specificp67 Ongoing

T. orientalis Beta-tubulin Ongoing

7.T. cruzi 18s rRNA Specific8.T. rangeli 5S rRNA Ongoing

5.T. evansi PFRA UniversalRoTat 1.2 VS Specific

6.T. equiperdum PFRA UniversalRoTat 1.2 VSG Specific

Andy Alhassan, Oriel M. M. Thekisoe, Naoaki Yokoyama, Noboru Inoue, Makhosazana Y. Motloang, Peter A. Mbati, Hong Yin, Chihiro Sugimoto, Ikuo Igarashi

Vet. Parasitol. In press (2006)

Development of LoopDevelopment of Loop--Mediated Isothermal Mediated Isothermal Amplification (LAMP) MethodAmplification (LAMP) Method for Dfor Diagnosis iagnosis

of Equineof Equine piroplasmosispiroplasmosis

Development of LAMP for specific diagnosisDevelopment of LAMP for specific diagnosis of of T. equi T. equi (B. equi)(B. equi) and and B. caballiB. caballi

ObjectiveObjective

T. equi (B. equi) B. caballi

LAMP primers for the amplification of LAMP primers for the amplification of T. equiT. equi and and B. caballiB. caballi

ParasiteParasite Target geneTarget gene SequenceSequence

Theileria equiTheileria equi EMAEMA--11F3: TTGCCATTTCGAGCATCCTF3: TTGCCATTTCGAGCATCCTB3: ACGGTCTTTGGGGTATGTTCB3: ACGGTCTTTGGGGTATGTTCFIP: TGCTTGTCGATGGTGATGTGGTFIP: TGCTTGTCGATGGTGATGTGGT--AGGAGGAGAAACCCAAGGCAGGAGGAGAAACCCAAGGCBIP: GTCCGAGGAGCACGTCGTCTABIP: GTCCGAGGAGCACGTCGTCTA--GTCTTGATGACGGAGTCGCGTCTTGATGACGGAGTCGC

BC48BC48F3: TGTTTCCATCATGGCTCCCF3: TGTTTCCATCATGGCTCCCB3: GCGCTAACGGAAGCACTGB3: GCGCTAACGGAAGCACTGFIB:GGCATTGGCAGCTGAGTCCAFIB:GGCATTGGCAGCTGAGTCCA--GGCGACGTGACTAAGACCTGGCGACGTGACTAAGACCTBIP:AGCGATTACTTGTCGGCTGTGTBIP:AGCGATTACTTGTCGGCTGTGT--CCCCTTAGGGACCTGACTGCCCCTTAGGGACCTGACTG

Babesia caballiBabesia caballi

Specificity of LAMP primersSpecificity of LAMP primers

MM RR TeqTeq BcBc BbBb TTevev MM TeqTeqBcBc BbBb TTevev RR

B. caballiB. caballi LAMP primersLAMP primersT. equi LAMP T. equi LAMP primers primers

M: 100 bp DNA ladder, R: horse RBCTeq: Theileria equi, Bc: Babesia caballi, Bb: B. bovis, Tev: Trypanosoma evansi

PCR with F3 and B3 LAMP primers PCR with F3 and B3 LAMP primers

T. equiT. equi primers B3 and F3primers B3 and F3 B. caballiB. caballi primers B3 and F3primers B3 and F3

MM RR TeqTeq BcBc BbBb

M: 100 bp DNA ladder, R: horse RBCTeq: Theileria equi, Bc: Babesia caballi, Bb: B. bovis

MM RR TeqTeq BcBc BbBb

002244

10101818262634343535

Days post Days post inoculationinoculation ELISAELISA LAMP LAMP

------ ++

-- ++++ ++++ ++++ ++++ ++++ ++

MicroscopyMicroscopy

----

++++++++----

PCRPCR

--++

++++++++++++

Detection of Detection of T. equiT. equi infection by Microscopy, ELISA, infection by Microscopy, ELISA, PCR and LAMP in experimentally infected horsePCR and LAMP in experimentally infected horse

T. equiT. equi B. caballiB. caballiNo. of blood No. of blood samplessamples

No. of positive (%)No. of positive (%)

mixed mixed infectioninfection

ChinaChina

South AfricaSouth Africa 3737

5555 45 (81.8 )45 (81.8 ) 31 (56.3)31 (56.3) 27 (49.0)27 (49.0)

34 (91.8 )34 (91.8 ) 17 (45.9)17 (45.9) 15 (40.5)15 (40.5)

CountryCountry

Application of LAMP for diagnosis of equine Application of LAMP for diagnosis of equine piroplasmosis on field samplespiroplasmosis on field samples

ConclusionsConclusions

LAMP specifically amplifies T. equi EMA1 gene and B. caballi Bc48 genes.

These LAMP primers are highly sensitive.

LAMP detected both T. equi and B. caballi, including mixed infections in blood samples collected from China and South Africa.

Further improvements and large scale evaluation are necessary for future use.

National Research Center for Protozoan Diseases

Research Unit for Advanced Preventive Medicine