Development of real-time polymerase chain reaction assay with TaqMan probe for the quantitative detection of infectious hypodermal and hematopoietic necrosis virus from shrimp.
An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies, with a dynamic range of detection between 9 x 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-react with shrimp genomic DNA or other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.
Yue
Zhi-Qin
ZQ
Shandong Entry-Exit Inspection and Quarantine Bureau, Food Admission Section, No. 1, Tuandao Er Rd, Qingdao 266002, People's Republic of China. yuezhiqin@126.com
Liu
Hong
H
Wang
Weiji
W
Lei
Zhi-Wen
ZW
Liang
Cheng-Zhu
CZ
Jiang
Yu-Lin
YL
D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003445:Crustacea / Q000821:virology*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D029142:Infectious hematopoietic necrosis virus / Q000378:metabolism*; D009336:Necrosis; D015345:Oligonucleotide Probes / Q000032:analysis*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load; D014777:Virus Diseases / Q000378:metabolism
D017931:DNA Primers; D015345:Oligonucleotide Probes; D004247:DNA