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Progress on the LAMP assay

for TB diagnosis

Tetsu Hase, Hidetoshi Kanda, Tsugunori Notomi

and Junji Morikawa (Eiken Chemical Co., Ltd.)

2(11)

F1c

F1c

F1c

F1

F1

F2

F2

F2cB1

B1

B1c

B1c

B1c

B2

B2B2c

FIP FIP

BIP BIP

Target DNA5'3'5' 3'

5'3'

F1cF2cF3c B1 B2 B3

B1c B2c B3cF1F2F3

5' F3 3'

3'B2

B1c 5'

5'3' B3

FIP

F3primer

BIP

B3primer

F1cF2

・DNA polymerase （with strand displacement

activity）・dNTPs・Mg++

Incubate at 60℃～65℃

Mechanism of LAMP reaction

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LAMPamplification

( TB )

Calcein

dNTPs

Mn2+

UV

quenching

Calcein

PPi + Mn2+

Mn2 P2 O7

UV fluorescence

DetectionDetection - visible fluorescence -

＋-

* No need of special equipment for the detection (only UV lamp is required).

* It enables to avoid cross contamination since it is performed in a closed tube from amplification to detection.

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Procedure of the LAMP method

Target DNAPrimers （FIP, F3, BIP, B3）DNA Polymerase with strand displacement activitydNTPsBuffer solution

60 – 65C15 - 60 min.

Detection

SimpleRapid (high efficiency)Specific (high specificity)Cost effective

Procedure of LAMP method

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Development of LAMP assay for detection of

Mycobacterium tuberculosis complex (TB-LAMP)

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TB-LAMP Assay

(C. Boehme et. al., J. Clin. Microbiol. 2007 45: 1936-1940. )

Detection by naked eye

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N N P

18

1 2 3 4

5 6 7 8 10 11 12

13 14 15 16 17

P

19 20

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Specimen Smear Amplicor LAMPNo. result result result

1 － TB ＋2 － TB ＋3 G1 TB －4 － TB －5 － TB ＋6 G3 TB ＋7 G8 TB ＋8 － TB ＋9 － TB ＋

10 G1 TB ＋11 G1 TB ＋12 G1 TB ＋13 － TB ＋14 G1 TB ＋15 G1 TB ＋16 G2 TB ＋17 － TB ＋18 G2 TB ＋19 G1 TB ＋20 G5 TB ＋NC －PC ＋

**

*; Small specimen volume (50%)

TB-positive specimens

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Specimen Smear Amplicor LAMPNo. result result result

1 G3 M. avium －2 － M. avium －3 － M. avium －4 － M. avium －5 G1 M. avium －6 － M. avium －7 － M. avium －8 G1 M. intraceluurae －9 － M. intraceluurae －

10 G6 M. intraceluurae －11 － － －12 － － －13 － － －14 － － －15 － － －16 － － －17 － － －18 － － －19 － － －20 － － －21 － － －22 － － －23 － － －24 － － －25 － － －NC －PC ＋

N N P 1 2 3 4P

5 6 7 8 10 11 129

1813 14 15 16 17 19 20

21 22 23 24 25

TB-negative specimens

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Lima, Peru(Site: University Cayetano

Heredia) Mbeya, Tanzania(Site: Mbeya

Medical Research Program)

Dhaka, Bangladesh(Site: ICDDR [International Centre for Diarrhoeal

Disease Research])

TB-LAMP Feasibility Study(Conducted by FIND-EIKEN; Feb. –

May, 2006)

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1) Clinical performance

a) Sensitivity (in smear +, LJ +) : 95.5-99.9% (av. 97.7%; 173/177)

b) Sensitivity (in smear -, LJ +): 32.0-63.0% (av. 47.5%; 19/40)

c) Specificity (in smear -, LJ -): 98.1-99.9% (av. 99.0%; 509/514)

2) The hands-on-time for the testing was similar to that of sputum smear microscopy.

3) No indeterminate results were reported and the inter-reader variability was 0.4%.

4) Despite the use of a simple room for all procedures, no DNA contamination was observed.

5) The assay was robust with high end-point stability and low rate of test failure.

6) Technologists with no prior molecular experience were able to easily perform the assay.

Results of the TB-LAMP Feasibility Study

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Current TB-LAMP assay is:<Usability>・Rapid:

* Results can be obtained within 100 min. including the DNA extraction. ・Easy-to-use:

* Entire procedure is very simple and easy to operate. * It does not require any sophisticated equipment. * However simpler assay with less steps is desirable.

< Performance＞・Specific:

* It detects TB complex specifically (no cross reaction with NTB).・Sensitive:

* Detection rate of smear positive sample is high.* Higher sensitivity is required for the detection of smear negative,

culture positive sample.

Now we are focusing on the improvement of TB-LAMP assay to be

more sensitive with a simpler operation.

12Thank you for your kind attention.

Acknowledgments

FIND

Dr. G. Roscigno

Dr. M. Perkins

Dr. C. Boehme

Kobe Institute of Health, Japan

Dr. M. Ito

Dr. T. Iwamoto

Feasibility study sites

* Dr. M. Hoelscher (U. Munich, Germany)

* Dr. P. Nabeta , Dr. G. Henostroza

and Dr. E. Gotuzzo

(U. Peruana ayetano Heredia, Peru)

* Dr. R. Raqid (ICDDR, Bangladesh)

EIKEN will continue to contribute to the health of people

as a general manufacturer of clinical diagnostics.