Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification(February 2014)

Chuanfeng Li, Zongyan Chen, Chunchun Meng, Guangqing Liu

INTRODUCTION

INTRODUCTION

HEPATITIS

Hepatitis is an inflammatory disease that affects the liver, this is generated infectious , Immune or toxic reaction . It is also considered, depending on its etiology, a sexually transmitted disease.

HEPATITIS A

Hepatitis A is a viral liver disease that can cause mild to severe symptoms

The hepatitis A virus is transmitted by ingestion of contaminated food or beverages or by direct contact with a person infected with the virus, Is associated with lack of clean water and poor sanitation

.

RT-PCR

•Amplification of RNA through previous synthesis of DNA copy, using a reverse transcriptase, followed by several cycles of conventional PCR, this technique can determine the expression of genes in different tissues

OBJECTIVE

Evaluate the specificity and sensitivity in the conserved regions of 3D gene of duck hepatitis A virus genotype C (DHAV – C), using reverse transcription loop-mediated isothermal amplification (RT-LAMP), for the prevention and rapid disease diagnosis.

MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

• DISEÑO PRIMER´S

Tabla 1. Detalles de RT-LAMP y RT-PCR primers diseñados para la detección de DHAV-C GX1201 cepa.

MATERIALES Y MÉTODOS

• EXTRACCION DEL RNA Y DNA

• RNATemperatura:-80

Temperatura:-20 ° C

RT-LAMP

RT-PCR

MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

RESULTADOS

Fig 1. Determinación de las condiciones óptimas para el ensayo de RT -LAMP

RESULTADOS

Fig 2. Detección de productos de RT-LAMP por métodos alternativos.

RESULTADOS

Fig 3. Especificidad del ensayo de RT-LAMP y RT-PCR.

RESULTADOS

Tabla 2. Comparación de ensayo de RT-LAMP y RT-PCR en muestras clínicas.

DISCUSSION

CONCLUSIONS

• The detection limit of RT-LAMP was about 100 times higher than of the RT-PCR, which suggests that the RT-LAMP was more sensitive than RT-PCR.

CONCLUSIONS

• The RT-LAMP is a simple technique of early diagnosis, rapid, highly specific, sensitive and cost effective for the detection and monitoring of potential infection DHAV-C.

• Primer design is crucial for the differentiation of DHAV-C from other genotypes or the specificity of this assay.

CONCLUSIONS

• The RT-LAMP can be widely used in the field or laboratory with limited resources, because their results can be easily detected with the naked eye due to increased turbidity or discoloration.

Manuela Diaz Valenzuela

Juliana Caro Palacio

THANKS