Group 4_LAMP Assay

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MOLECULAR BIOTECHNOLOGY

LECTURER: DR. TRAN NGOC DUC

GROUP 4

1. TONG NGUYEN NHAT ANH

2. TRAN THAI MY DUYEN

3. TRINH HAI MY4. NGUYEN NGOC THIEN HUONG

5. TRUONG LE PHUC NHI

6. NGUYEN HAI QUAN

7. NGUYEN HUU THOI


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OUTLINE

I INTRODUCTION

II MATERIALS AND METHODS

III RESULT

IV

VALIDATION

V CONCLUSION


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Bacterial meningitis?

Meningitis: an infection of the

membranes (meninges) surrounding the

brain and spinal cord

Caused by a bacterial, fungal or viralinfection.

I. Introduction

Bacterial meningitis: caused by one or several types of

bacteria(Haemophilus influenzae type b, Neisseria meningitidis, and Streptococcus

pneumoniae)

The infection can cause the tissues around the brain to swell ->

interferes with blood flow -> result in paralysis or even stroke.


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Conventional diagnosis of bacterial meningitis

Method Advantages LimitationGram staining Quick Low sensitivity

Culturing Takes few days

PCR High sensitivity

& specificity

Expensive instrument

Experienced technicianFew-hour performance

Loop-mediated isothermal amplification (LAMP) assay

Accurate

Rapid

Cost-effective method


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Aim

Design and develop LAMP assay capable of detecting multiple

bacterial species based on the nucleotide sequences of the 16S

rRNA genes of the bacteria

(Staphylococcus aureus , Streptococcus pneumoniae, S. suis andS. agalactiae )


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II. Materials and methods

1. LAMP assay

LAMP = loop-mediated isothermal amplification( Isothermal amplification process using a strand displacement reaction)

Characterized by the use of 4 different primersspecificallydesigned to recognize 6 distinct regions of the target gene


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Stages in Loop-mediated Isothermal Amplification

Production of starting material

Cycling amplification with elongation

Recycling


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2. Materials

Bacterial strains

Serotypes 3 and 10 of S. pneumoniae isolated from upper

respiratory tract in Vietnamese patients

Strains 8-01 and 8-02 of S. suis serotype 2E. coli, S. aureusand S. agalactiae also isolated from Vietnamese

patients

H. Influenzae, N. meningitidis isolated from Japanese patients


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Samples preparation

Grow bacteriaon agar

Harvested &suspended innormal saline

Collect cells

Suspend cells inTE buffer

Dilute with TEbuffer

Boiling (20 min)

Centrifuge

(10,000 rpm, 10min)

Get supernatant


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3. Methods

Design of LAMP assay primers

The broad range LAMP primers were designed to be specific

for bacterial 16S rRNA-specific gene


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LAMP primer sequences for simultaneous detection of

S. pneumoniae , S. agalactiae , S. aureus andS. suis


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Optimization of LAMP reaction

Loopamp DNA amplification kitPrimers FIP, BIP 40 pmol (1 uL)/each

F3, B3 5 pmol (1 uL/each)

Loop F, loop B 20 pmol (1 uL/each)

Extracted DNA 2 uL

Positive control a confirmed positive sample

Negative control a reaction mixture withdistilled water instead of

DNA template

Total amount 25 uL


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LAMP reaction was performed at

several different temperatures ranging

from 55 to 68 C in 90 min using LA-

320C Loopamp real-time turbidimeter.

The best condition for LAMPprocedure was at 63 C and in 60 min.

Therefore, all of mixtures were

incubated at 63 C for 90 min,

followed by heating at 80 C for 5 minto inactivate the reaction.


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After amplification:

2 uL of the LAMP product was further separated by 2%

agarose gel electrophoresis.

The remained LAMP product: added 1 uL of SYBR Green I

A change from orange to fluorescent green col-our: positive

To distinguish bacterial species: digested 2 uL of the LAMP

product with 10 U of DdeI or HaeIII at 37C for 90 min.

The digested LAMP product was analysed by 2% agarose gel

electrophoresis


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III. Result

Visual detection of the LAMP products

Visual inspection of S.pneumoniaeby fluorescence

Under ultraviolet lamp Under day light


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Identification of bacterial species

HaeIII digestion patternsDdeI digestion patterns


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IV. Validation

Sensitivity of LAMP assay

Serially dilute 10 - fold of target bacteria(from 10*7 to 10*0 CFU mL*-1)

Detection of S. pneumoniae Sensitivities of LAMP and conventional PCRassays

The sensitivity of LAMP assay was 100 1000 times higher

compared with the conventional PCR assay


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Specificity of LAMP assay

A mixture of DNA template extracted from:

4 species for detection Other bacteria

- Staphylococcus aureus

- Streptococcus pneumoniaeserotypes 3 & 10

- Streptococcus suis serotypes 2-

8-01 & 2-8-02

- Streptococcus agalactiae

- Haemophilus influenzae

- Escherichia coli- Neisseria meningitidis


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Specificity of broad range LAMP assay


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V. Conclusion

Four common pathogen of BM including S. pneumoniae,

S. suis, S. agalactiae andS. aureus could be detected using

LAMP assay

Advantages Limitation

- Rapid (< 1h)

- Simple

- NOT require highly experienced

technician- Can be performed in a water

bath at bedside or in rural areas

Only four species could be

detected


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THANK YOU FOR KIND

ATTENTION!

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Group 4_LAMP Assay

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