Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China.
A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.
Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Xing-Juan
XJ
Zhang
Qiu-Ying
QY
Han
Yuan
Y
Sun
Sándor
S
Belák
Lihong
L
Liu
Hua-Ji
HJ
Qiu
D000818:Animals; D052160:Benzothiazoles; D002681:China; D006691:Classical Swine Fever / Q000175:diagnosis* / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D003937:Diagnosis, Differential; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D013696:Temperature; D014765:Viral Vaccines; D014773:Virology / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D014765:Viral Vaccines; C098022:SYBR Green I