Detection Of Shrimp White Spot Syndrome Virus By Loop-mediated Isothermal Amplification Combined With A Lateral Flow Dipstick

In this study, we had developed a White spot syndrome virus (WSSV) diagnostic procedure called Loop-mediated isothermal amplification (LAMP) and conbined it with FITC nucleic acid detection lateral flow dipstick (FITC-LFD), called LAMP-LFD, which is an effective and economical assay with high sensitivity, more specificity, and is very fitable for clinic diagnosis.According the LAMP principle, a set of four specific primers were designed by Primer Explorer V4, based on the conservative gene of WSSV envelope protein 28 (VP28), published in GenBank. Using the recombinant plasmid of pMDT-VP28 as a standard template, the LAMP method for WSSV detection was developed by optimizing its reaction system and conditions. Its detection limit for WSSV was then determined by detecting the ten-fold diluted WSSV-DNA (107-10-3 copies/μL) and compared with nested polymerase chain reaction (nested-PCR). The reaction temperature and time of WSSV LAMP were optimized at 65℃for 60 min, respectively. Detection limit(DL) of the LAMP method for the WSSV-DNA was determine to be 10 copies/μL, whereas that of nested-PCR was 100 copies/μL, suggesting the LAMP method was much more sensitive than the PCR methods for WSSV detection.At the same time, on the basis of affinity between fluorescein isothicyanate (FITC) and protein, Two complete antigens of FITC, called FITC-BSA and FITC-OVA, were prepared; Then the compound's characterization was identified by UV(ultraviolet spectrum) and SDS-PAGE, respectively. BALB/c mice were immunized with FITC-BSA, the titre of polyclonal antibody (pAb) was detected by indirect ELISA, the sensitivity and specificity of pAb was identified by blocking ELISA. FITC mAb was prepared by hybridoma technology. Then the titer, sensitivity and specificity of the mAb were characterized. Massive FITC mAb were induced from in vivo method. The results of UV spectrum and SDS-PAGE suggested that the conjugation was occur and successful. The results of indirect ELISA and indirect competent ELISA showed the titre of pAb for 1# mouse was the highest and IC50 was the lowest.1# mouse was chosen for cell fusion, and six highly sensitive and specific hybridomas were obtained after screening. After identified by ELISA,1B11 was used to produce ascites. The titre of FITC-mAb for 1B11 was 1/640000, its IC50 was 7.812ng/mL.Following the LAMP-LFD protocol, the FITC-mAbs, labelled colloidal gold, were fixed on glass cellulose film 3μL/stick.0.5mg/mL avidin and 1mg/mL GaMIgG-HRP were fixed on test line and control line,1.2μL/cm respectively, on Nitrocellulose membrane. After assemble FITC nucleic acid detection Lateral flow dipstick, we designed a special FIP with biotin at 5'end and a probe with FITC at 5' end. The LAMP-LFD reaction conditions were optimized before detection limit for this method, determined by detecting the ten-fold diluted WSSV-DNA (107-103 copies/μL). The reaction temperature and time of WSSV LAMP were optimized at 64℃for 50 min, respectively. Detection limit (DL) of the WSSV-LAMP method for the WSSV-DNA was determine to be 10 copies/μL, as the same as LAMP's.