Rapid detection of BoHV-1 genomic DNA by loop-mediated isothermal amplification assay.
Bovine herpes virus-1 (BoHV-1) is a serious viral pathogen of domestic and wild cattle. Herein, we report development of a new molecular diagnostic assay for rapid and sensitive detection of BoHV-1 utilizing the loop-mediated isothermal amplification (LAMP) technique. BoHV-1-LAMP assay was optimized to amplify the target DNA by incubation the Bst-DNA polymerase enzyme with a set of specially constructed six primers, based on the gE-gene of BoHV-1 virus, at 65°C for 60min. BoHV-1-LAMP products were detected by visual inspection using SYBR Green-I stain and had a ladder-like appearance by gel electrophoresis analysis. Negative results obtained with DNA from other tested fish viruses confirmed the specificity of the assay. The analytical sensitivity of the BoHV-1-LAMP assay was 1fg of BoHV-1 DNA (dilution of 10(6)). The developed assay could successfully detect BoVH-1 DNA from clinical samples. Results of this study indicate that the developed BoHV-1-LAMP is rapid and highly sensitive assay not only for detection of BoHV-1 in clinical samples, but also for differentiation between wild-type (gE-positive) and gE-negative BoHV-1 viruses, which will improve the control programs of BoHV-1 in Egypt.
Veterinary Serum & Vaccine Research Institute, Abbassia, P.O. Box # 131, Cairo, Egypt.
Alaa A
AA
El-Kholy
Parasitology and Animal Diseases Department, National Research Center, Dokki, Giza, Egypt.
Khaled
K
Abdelrahman
Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, 71515 Assiut, Egypt. Electronic address: hatemtoughan@hotmail.com.
Hatem
H
Soliman
D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology*; D017931:DNA Primers / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003959:Diamines; D004534:Egypt; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000032:analysis; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D013995:Time; D014730:Veterinary Medicine / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004259:DNA-Directed DNA Polymerase
Abortions; BoHV-1 gE-gene; LAMP; Pneumoenteritis; SYBR Green-I stain