Applications of competitor RNA in diagnostic reverse transcription-PCR

Abstract

Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

FIG. 1.

Method for preparation of in vitro-transcribed RNA containing PRRSV viral primer sequences. (A) Schematic of the relative oligonucleotide primer sequence positions, synthesis steps, and time required for each step. (B) Approximately 1 μg of in vitro-transcribed RNA (lane 1) following denaturing agarose gel electrophoresis with ethidium bromide staining. Lane M, RNA molecular size markers.

FIG. 2.

Detection of serial 10-fold dilutions of in vitro-transcribed RNA by PRRSV RT-PCR. RT-PCR (lanes 1 to 6) or PCR (lanes 7 to 12) was performed with serial dilutions of in vitro-transcribed RNA containing approximately 20,000 (lanes 1 and 7), 2,000 (lanes 2 and 8), 200 (lanes 3 and 9), or 20 (lanes 4 and 10) RNA molecules per μl. The results obtained following agarose gel electrophoresis are shown. The results for negative (no template added) controls are shown in lanes 5 and 11. The results for positive control reactions in which the template was PRRSV RNA (lane 6) and a previously amplified RNA mimic (lane 12) are also shown. Lane M, DNA molecular size markers.

FIG. 3.

Use of RNA mimics as extraction controls. PRRSV RT-PCR was performed with serial dilutions of in vitro-transcribed RNA either before (lanes 2, 4, and 6) or after (lanes 1, 3, and 5) column extraction of samples containing 50 (lanes 1 and 2), 500 (lanes 3 and 4), or 5,000 (lanes 5 and 6) RNA molecules per μl. Lanes 7 and 8, results for negative (no template) and positive (PRRSV RNA) control reactions, respectively; lane M, DNA molecular size markers.

FIG. 4.

Detection of endogenous RT-PCR inhibitors in samples submitted for diagnostic assays. Prior to PRRSV RT-PCR, 100 molecules of competitor RNA were added to RNA extracted from six swine serum samples. The results of PRRSV RT-PCR following agarose gel electrophoresis for samples from which viral RNA was extracted with Trizol (lanes 1 to 6) or Qiagen (lanes 7 to 12) columns are shown. The results for negative (no template) and positive (PRRSV RNA) control reactions are shown in lanes 13 and 14, respectively. Lane M, DNA molecular size markers.

FIG. 5.

Detection of endogenous RT-PCR inhibitors in a sample used for diagnosis. Prior to WNV RT-PCR, serial dilutions of competitor RNA were added to undiluted and diluted (1:20) RNA extracted from a spleen sample. The results of RT-PCR following agarose gel electrophoresis are shown. Lanes 1 to 7, 10, 100, 1,000, 10,000, 100,000, 1,000,000, and 0 molecules of competitor RNA, respectively; lane M, DNA molecular size markers.

FIG. 6.

Detection of endogenous RT-PCR inhibitors in samples used for diagnosis analyzed by 5′ exonuclease (TaqMan) RT-PCR. Two dual-labeled probes, one for detection of the WNV RNA amplification product and one for detection of the heterologous competitor RNA amplification product, were included in each reaction mixture. Amplification was performed with reaction mixtures to which 20 copies of competitor RNA were added to each mixture. The horizontal line at 0.01 fluorescence units indicates the threshold for a positive reaction. dRn, baseline-corrected normalized fluorescence.

FIG. 7.

Quantification of PRRSV RNA in serum samples. Prior to RT-PCR, dilutions of the competitor RNA were added to RNA extracted from serum from PRRSV-infected pigs. Equal amounts of RNA purified from serum were mixed with increasing amounts of competitor RNA. The results of RT-PCR following agarose gel electrophoresis are shown. Lanes 1 to 7, 0, 10, 50, 100, 500, 1,000, and 5,000 molecules of competitor RNA, respectively; lanes 8 and 9, results for negative (no template) and positive (PRRSV RNA) control reactions, respectively; lane M, DNA molecular size markers.