DETECTION OF SHRIMP TAURA SYNDROME VIRUS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION USING A DESIGNED MULTICHANNEL PORTABLE TURBIDIMETER

Wansadaj Jaroenram*, Assawapong Sappat, Wansika Kiatpathomchai, Tanom Lomas, Adisorn Tuantranont and Timothy Flegel


CENTEX Shrimp and Department of Biotechnology, Faculty of Science, Mahidol University,
Rama 6 Road, Ratchathewi, Bangkok 10400 Thailand. Email kungbtram@gmail.com Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the synthesis of large amounts of DNA in a short time with high specificity. Since a white magnesium pyrophosphate (Mg2P2O7) precipitate is a characteristic by-product of LAMP reactions, a simple turbidimetric, end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) by spectroscopic measurement of the LAMP reaction precipitate. The device incorporated a heating block that maintained the optimal temperature of 63for the duration of the 25 LAMP reaction performed in a 0.2 ml tube. The temperature control and the turbidity measurements of this apparatus were sufficiently uniform for conducting LAMP reactions and monitoring the turbidity. The optimal conditions for TSV-LAMP was 6330 min. Using these conditions, LAMP-turbidity measurement revealed comparable sensitivity to that of LAMP-AGE, LAMP-LFD, nested PCR but showed 100 times greater than one step PCR. Cross reactions with other shrimp viruses as templates was not found, indicating that the LAMP methods were highly specific to TSV. Combining 10 min for nucleic acid preparation by a rapid method with 30 min for LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1 h compared to 4-8 h for the nested PCR. In addition, use of the turbidimeter yielded results immediately at the end of the LAMP reaction, without the need to open the reaction tube (i.e., avoidance of contamination) or to add further reagents. Thus, LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of shrimp viruses in the field without risk of amplicon contamination.

HAIRPIN-RNA EXPRESSION CASSETTE BY TWO-STEP CLONING SYSTEM AND ITS POTENTIAL AS AN IMMUNE STIMULANT IN SHRIMP

Vanvimon Saksmerprome*, Patai Charoonart, Boonsirm Withyachumnarnkul

National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani and Centex Shrimp, Mahidol University, Bangkok Thailand
E-mail: vanvimon.sak@biotec.or.th We demonstrate an improved method for delivering RNAi-based immunity to large shrimp populations. Long sequences of double-stranded RNA (dsRNA) have recently been used to enhance viral resistance, through an RNA interference (RNAi) mechanism, in shrimp aquaculture. Since dsRNA-mediated knockdown efficiency of viral genes appears to be dose dependent, a large-scale production of dsRNA is necessary for antiviral/therapeutic applications of RNAi for shrimp farm operations. A new design of hairpin-RNA expression vector, followed by transformation into RNase-deficient E.coli HT115, offers a quick preparation of a large amount of long dsRNA (normally >300 nt). The hairpin RNA consists of a forward strand and a 100-base shortened reverse strand, and the unpaired 100-base region on the forward strand serves as a loop. This strategy reduces 3-step to 2-step cloning of hairpin construct into DNA expression cassette, thus bypassing difficulty in joining a small loop piece into a large carrier vector. A total RNA of ~4 mg is generally obtained from 100 mL bacterial culture.

The bacterially expressed hairpin RNA specific to RNA-dependent RNA polymerase (RdRp) gene of yellowhead virus (YHV) can induce antiviral immunity in Penaeus vannamei shrimp. Viral protection by exogenous dsRNA demonstrates the potential of dsRNA produced by 2-step-cloning hairpin cassette as an immune stimulant in shrimp (Figure 1). We investigate further if the putative viral promoters can be regulated by shrimp transcriptional activators. The hairpin specific to YHV is cloned into the viral promoter-contained plasmids, followed by intramuscular injection of the constructs into shrimp. We examine protection against YHV and level of viral mRNA after plasmid injection. Successful hairpin expression driven by such promoters demonstrates the potential of DNA-based vaccine for effective antiviral immunity in shrimp.