| Chicken infectious anemia virus (CIAV) is a virus that will cause the atrophy of bone marrow hematopoietic and lymphoid tissues in young chickens and lead to aplastic anemia which named Chicken infectious anemia(CIA).The infection has been the most common immunosuppressive diseases in animal husbandry around the wirld.It usually merge,secondary and aggravate other virus,bacteria and fungal infections in the clinical,leading to the occur of mixed infection.It will also seriously affect the growth of the chicks.To be as an infectious disease,the CIAV has caused serious damage to Chinese poultry industry,which need us to pay more attention.So establishing an accurate and rapid diagnostic method is the precondition for the prevention and control of the disease,it has important clinical significance.This study is established on LAMP detection methods,a emerging technology,perfecting this method continuously,so as to explore a method which can widely used to CIAV detection and eventually make a convenient,fast and cheaper way to the CIAV.The results were as following:1. This study used hydroxy bromophenol blue (HNB) as dye for the LAMP reaction.The positive and negative sample can be judged just with the naked eye.This way has strong visibility and everyone fell free to work.2. Four specific primers for CIAV were designed for the relatively conservative region of VP2 gene of CIAV using Blast,Meg Align and DNA Star.On the basic of the LAMP detection method,conducting continuous explorations and conditions optimization,establishing a sensitive and specific system of LAMP.The system was as following:In a 25?l total reaction mixturejoined 240?mol/L HNB and then led to a good result after LAMP reaction;To adjust the concentration of Mg,0.4 mmol/L Mg was perfect to get a good chromogenic result;Then,optimize the temperature,results showed that the LAMP primers which designed for this study can achieve wide range of temperature,CIAV all can effectively detected between 55?65?,which was very useful to take advantage of the inaccurate tempreture control equipment,such as Water Bath,and so on.Combining the result of Agarose gel,the optimal reaction temperature for the LAMP was 61?;When time set range from 20 min to 70 min,50 min can be the most efficient way to detect CIAV,thus the optimum reaction time was 50 min;when the Internal primer concentration was set as 0.6 ?mol/L in the system,a good result would be obtained.Through the optimization experiments,a optimal LAMP reaction system for the detection of CIAV has been built.3. Based on the constructed cloning vector of CIAV gene,ten-times step dilution the cloning vector of CIAV gene to test the sensitivity of the LAMP detection method.Reaction products then were detected by eyes and gel respectively,and compared with PCR reaction for the detection of the sensitivity of LAMP,the results showed that LAMP was ten times the sensitivity of common PCR.To Extract fowl adenovirus group l,egg drop syndrome virus,chicken infectious laryngotracheitis virus,Marek's disease virus,Mycoplasma Galliscepticum DNA,CIAV positive sample and normal chicken tissue DNA,LAMP was used to inspect their specificity.The results showed that LAMP can just detect CIAV.To detect 20 clinical samples,the relevance ratio was 100%,the repeated test results were basically identical,showing that LAMP is an appropriate method for CIAV.This study can set up a foundation for the development of CIAV detection kit.It has the certain promotion application value. |
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