Application of high resolution melt (HRM) analysis for duplex detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in shrimp.
In this work, a probe-free, multiplex RT-PCR was combined with high resolution melt (HRM) analysis for the simultaneous detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) infection in the freshwater prawn Macrobrachium rosenbergii. This first application of HRM multiplex RT-PCR in shrimp reveals a new potential for rapid and sensitive detection of multiple pathogens. In addition, sequence variation in XSV could be observed from the high resolution melt peaks, as confirmed by sequence analysis. In 19 field samples of the freshwater prawn M. rosenbergii the technique revealed samples negative for both viruses, positive for both viruses or positive for MrNV alone. No sample was found positive for XSV alone. Comparison of these results to those obtained using the same samples in analysis by traditional nested RT-PCR combined with gel electrophoresis revealed that HRM multiplex RT-PCR was more sensitive. Thus, the latter technique allows for rapid and sensitive, simultaneous detection of MrNV and XSV and also has the potential to be adapted for simultaneous detection of other mixed viral infections in shrimp.
Senapin
Saengchan
S
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand. saengchan@biotec.or.th
Molthathong
Sudkhate
S
Phiwsaiya
Kornsunee
K
Jaengsanong
Chatlada
C
Chuchird
Niti
N
D000818:Animals; D030381:Nodaviridae / Q000235:genetics* / Q000302:isolation & purification; D009691:Nucleic Acid Denaturation; D033541:Palaemonidae / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature; D014780:Viruses / Q000235:genetics* / Q000302:isolation & purification
D012367:RNA, Viral