Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification.
Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions for the LAMP assay were 60 min at 63 degrees C. The sensitivity of LAMP for PemoNPV detection was approximately 50 viral copies ng(-1) genomic DNA (equivalent to 150 viral copies per reaction). Using a DNA template extracted from PemoNPV-infected shrimp by a viral nucleic acid kit, the detection limit of LAMP was 0.7 fg while that of nested PCR was 70 fg; therefore, the LAMP assay was 100 times more sensitive than nested PCR. The LAMP method did not amplify a product using nucleic acid extracted from shrimp infected with other viruses including yellow head virus (YHV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV) known previously as infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Penaeus monodon densovirus (PmDNV) known previously as hepatopancreatic parvovirus (HPV).
Chaivisuthangkura
Parin
P
Department of Biology, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand. parin@swu.ac.th
Srisuk
Chutima
C
Rukpratanporn
Sombat
S
Longyant
Siwaporn
S
Sridulyakul
Pattarin
P
Sithigorngul
Paisarn
P
D000818:Animals; D017931:DNA Primers; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017924:Nucleopolyhedroviruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D000077183:Occlusion Body Matrix Proteins; D033561:Penaeidae / Q000821:virology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D000077183:Occlusion Body Matrix Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus