Saturday, July 31, 2010


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 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.

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