Development of internal controls for probe-based nucleic acid diagnostic assays.
A method is described for the design, evaluation, and application of internal control targets and probes for use in probe-based nucleic acid diagnostic assays (i.e., PCR-ELISA). The technique is a modified version of oligonucleotide-directed mutagenesis in conjunction with PCR amplification to develop a novel probe-annealing sequence in a cloned IS1111a gene fragment of Coxiella burnetii. The internal control probe-recognition site with its complementary probe was identical to the wild-type-specific probe in length, base composition, location, and annealing temperature. Neither the internal control nor the wild-type probes annealed to the recognition sequence of the other. As both of the amplified nucleic acid fragments, internal control and wild type, were identical in length and base composition, the amplification conditions for the diagnostic assay were not affected. This allowed small copy numbers of the internal control clone to be loaded into a diagnostic assay without negatively affecting it. In a single reaction we were able to differentiate between an assay reporting a true or false-negative signal. A negative signal is defined as the absence of detectable pathogen genetic material (true) or inhibition/failure of the reaction (false).
Courtney
B C
BC
Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA.
Smith
M M
MM
Henchal
E A
EA
D001483:Base Sequence; D016997:Coxiella burnetii / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics / Q000592:standards; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D005188:False Negative Reactions; D006801:Humans; D015336:Molecular Probe Techniques / Q000592:standards* / Q000706:statistics & numerical data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D011778:Q Fever / Q000175:diagnosis / Q000382:microbiology; D011786:Quality Control; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial