Development of a ssRNA internal control reagent for an infectious bursal disease virus reverse transcription/polymerase chain reaction-restriction fragment length polymorphism diagnostic assay.
Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens. A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay. An 841-bp bacteriophage-lambda DNA fragment was directionally ligated to 3' and 5' oligonucleotide linkers containing the IBDV RT/PCR target primer sequences. A pGEM-3Zf (+) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA. After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product. The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms. With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay. By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results.
Smiley
J R
JR
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA.
Sommer
S E
SE
Jackwood
D J
DJ
D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis* / Q000235:genetics; D002645:Chickens; D005188:False Negative Reactions; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000472:pathogenicity; D012150:Polymorphism, Restriction Fragment Length; D011201:Poultry Diseases / Q000175:diagnosis* / Q000235:genetics; D012313:RNA / Q000032:analysis / Q000235:genetics*; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*
D012313:RNA