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From the Clinical Virology Research Unit,
*
Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital and Health Service District; the Clinical Medical Virology Centre,
University of Queensland; the Microbiology Division,
Queensland Health Pathology Service, Royal Brisbane Hospital Campus; and the Department of Paediatrics and Child Health,
University of Queensland, Brisbane, Queensland, Australia
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Abstract |
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Introduction |
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Several studies have shown that polymerase chain reaction (PCR) amplification can resolve the intrinsic limitations associated with traditional diagnostic techniques by combining increased sensitivity, specificity, and rapid result turnaround time.16, 17 Also, PCR results are not dependent on infectious virus or viable cells. However, PCR may be affected by the presence of sequence variation that can be overcome by designing the assay to target highly conserved nucleic acid sequences.18 Also, using conventional PCR technology to detect several viruses individually is labor-intensive and expensive.18, 19 These limitations can be overcome by using a multiplex PCR assay. The multiplex format is a significant improvement over conventional PCR protocols, achieved by incorporating multiple primers that amplify RNA or DNA from several viruses simultaneously in a single reaction.9, 20, 21, 22, 23, 24, 25
In this study we combine the multiplex RT-PCR with an enzyme-linked amplicon hybridization assay (ELAHA) developed in our laboratory to detect amplification products using a colorimetric detection method.26 The ELAHA method can increase the sensitivity and specificity of PCR assays by detecting amplicon with a sequence-specific biotinylated probe.26, 27 We also incorporated an internal control PCR reaction. One of the major limitations of PCR detection is false-negative results as a consequence of PCR inhibitors present in the clinical sample that are not removed by the extraction process. The internal control PCR reaction incorporated in this multiplex RT-PCR, targeted sequences of the human endogenous retrovirus (ERV-3).28 To date, no published multiplex-PCR assay has been able to simultaneously detect adenovirus, flu A and B, RSV A and B, and Para 1, 2, and 3.
We evaluated the suitability of the m-RT-PCR-ELAHA as an alternative laboratory method to the traditional DFA or viral culture. In all, 598 nasopharyngeal aspirates from patients with suspected respiratory infection were tested for the presence of seven respiratory viruses. The results of the m-RT-PCR-ELAHA were compared to those obtained by a testing algorithm used in our hospital’s laboratory combining DFA and a culture augmented DFA (CA-DFA) method. Testing of a panel of DNA and RNA extracted from 41 unrelated organisms confirmed the specificity of the primers and probes used in the m-RT-PCR-ELAHA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extraction of Viral Nucleic Acids
Viral nucleic acids were extracted from 0.2 ml of each specimen in VTM using the High Pure Viral Nucleic Acid kit (Roche Diagnostics, Castle Hill, NSW, Australia), following the manufacturer’s instructions. Purified specimen nucleic acid was eluted from the column in 50 µl of kit elution buffer (Roche Diagnostics). Nucleic acid extracts were stored at –70°C until analysis and were used in the multiplex RT-PCR-ELAHA and the monospecific RT-PCR assays.
Multiplex Reverse Transcriptase PCR (m-RT-PCR)
The m-RT-PCR was performed using 0.2 ml thin-walled PCR tubes in a Perkin Elmer 9600 thermal cycler (Applied Biosystems, Foster City, CA, USA). The RT-PCR mixture used in the assay was the Superscript One-Step RT-PCR kit (with Platinum Taq) (Invitrogen, USA). Each reaction contained the following kit reagents: 1 µl of RT/Platinum Taq mix and 25 µl of 2X Reaction Mix (containing 0.4 mmol/L of each dNTP and 2.4 mmol/L MgSO4). To this were added four units of extra Platinum Taq (Invitrogen, Carlsbad, CA, USA) 10 pmoles of each primer (including the ERV primers to act as internal control; Table 1
), 0.1 nmoles of digoxigenin-11-dUTP (Roche Diagnostics) and 5 µl of specimen DNA or RNA extract or control. The final reaction volume was adjusted to 50 µl with PCR-grade water and RT-PCR amplification was performed using the following conditions: initial incubation for 30 minutes at 50°C for reverse transcription, followed by a denaturation step at 95°C for 2 minutes; followed by 45 cycles of denaturation at 95°C for 20 seconds; primer annealing at 58°C for 20 seconds; extension at 72°C for 20 seconds; and 1 cycle of further extension at 72°C for 7 minutes.
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using the protocol previously described.26
Briefly, following amplification, 5 µl of reaction mix was added to each of eight reaction tubes (200 µl) containing 10 ng of probe, specific for each of the seven respiratory viruses and the ERV, diluted in 5 µl of 1X sodium chloride/sodium citrate buffer (SSC) (3 mol/L sodium chloride, 0.3 mol/L sodium citrate). A 40-µl volume of 1X SSC was added to each tube giving a final reaction volume of 50 µl. The tubes were then placed into a thermocycler and held at 94°C for 5 minutes to denature the amplicon followed by rapid cooling to 4°C. The contents of the tubes were transferred to streptavidin-coated wells of a 96-well microtitre plate (Thermo-Labsystems, Ryolalmere, NSW, Australia) and incubated for 20 minutes at 37°C. The wells were washed four times with 1X SSC. Anti-digoxigenin peroxidase conjugate (Roche Diagnostics) was prepared by diluting the conjugate in phosphate-buffered saline (PBS, pH 7.2) to a final concentration of 150 mU/ml. A 100-µl volume was added to each well and incubated for 20 minutes at 37°C. Wells were again washed four times with 1X SSC. Finally, 100 µl of tetramethylbenzidine substrate (TMB, Elisa Systems, Windsor, Queensland, Australia) was added to each well and incubated at room temperature for 10 minutes. The reaction was stopped by the addition of 100 µl of 1 mol/L HCl. The optical density for each well was recorded using a plate spectrophotometer (Murex Diagnostics, Norcross, GA, USA) at a wavelength of 450 nm using 690 nm as a reference. An optical density of 0.2 or greater was indicative of a positive result.
Sensitivity of the Multiplex Reverse Transcriptase PCR
The sensitivity of the m-RT-PCR assay was determined for each of the targets using virus suspensions in cell culture media that had been quantified in terms of TCID50. The Tissue Culture Infectious Dose 50 (TCID50) is the quantity of virus in a specified suspension volume that will infect 50% of cell cultures. This was calculated for each virus suspension using the Karber method29
by inoculating a 10-fold dilution series of the original suspension onto cell monolayers cultured in the wells of a 96-well tissue culture plate (Nunc A/S, Kamstrup, Roskilde, Denmark). Each dilution was inoculated onto eight separate wells (cultures), and the number of ensuing infected monolayers was determined by direct fluorescent staining for each dilution.
To determine the sensitivity of the m-RT-PCR for each of the seven respiratory viruses (ADV, Flu A and B, PIV1, 2, 3, and RSV), six 10-fold dilutions in VTM were made of virus suspensions for which a TCID50 had previously been determined using standard virological technique.29, 30, 31, 32 Dilutions ranged from 5 x 104 to 0 TCID50 for each virus. Viral RNA or DNA was extracted from 0.2 ml of each dilution and tested by the m-RT-PCR assay using the conditions described above. Viral targets, for which the assay returned a sensitivity between 5 to 50 TCID50 per milliliter, were repeated using serially twofold dilutions ranging from 5 to 80 TCID50 per milliliter. This was done to determine a more accurate estimation of the analytical sensitivity in this range of viral concentrations. The highest dilution returning a positive RT-PCR result was considered to be representative of the assay’s analytical sensitivity.
Specificity of the m-RT-PCR
Specificity testing between the seven viral targets was performed using RNA and DNA extracted as detailed above from clinical isolates of Flu A, Flu B, PIV1, PIV2, PIV3, RSV types A and B, and Adenovirus. These isolates were subsequently used as the positive controls in each run. In addition, a panel of respiratory organisms found in the respiratory tract was used to determine the inter-specificity of the m-RT-PCR. This consisted of reference strains and clinical isolates shown in Table 2
. Nucleic acid from these organisms was purified and tested in the m-RT-PCR assay using the conditions described above. The amount of non-specific nucleic acid ranged from 0.4 to 0.9 µg per reaction.
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Culture Augmented-DFA
The culture augmented-DFA (CA-DFA) method was developed in our laboratory to replace viral isolation by cell culture. This method adopted the principles of a standard shell vial technique.15
Four continuous cell lines, MRC-5, A549, HEP-2, and LLC-MK2 (Biowhittaker, Walkersville, MD, USA), susceptible to respiratory virus infection were cultured in 96-well sterile tissue-culture plates (Nunc, Australia). An 80-µl volume of each NPA specimen was centrifuged onto the cell monolayers at 4000 rpm for 30 minutes at 35°C using a Hettich Rotanta 96R microtiter plate centrifuge (Hettich-Zentrifugen, Tuttlingen, Germany). Supernatants from each well were then aspirated and replaced with 200 µl of maintenance medium consisting of 194 µl of Eagle’s minimum essential medium (Biowhittaker) containing 2% (v/v) fetal bovine serum (Trace Biosciences, Sydney, NSW, Australia), 9.75 units of penicillin G (5000U/ml; CSL Biosciences, Australia), 10 µg of streptomycin sulfate (5000 µg/ml; CSL Biosciences, Melbourne, VIC, Australia), and 0.25 µg of fungizone (5 mg/ml; Apothecon, Princeton, NJ, USA). After 40 hours of incubation at 37°C in a 5% CO2 atmosphere, wells were aspirated and cell monolayers were fixed with an acetone/methanol mixture (1:1 v/v) for 10 minutes at –20°C. Cell monolayers in the wells were dried and then overlaid with 30 µl of appropriate monoclonal antibody (Intracel) specific for the target respiratory virus as described in the DFA method above. After incubation with anti-mouse FITC conjugate (Intracel), the presence of a respiratory virus was indicated by the detection of at least one single fluorescent cell under UV illumination using an inverted fluorescent microscope (Eclipse TE200; Nikon-Kawasaki, Kanagawa, Japan) at x100 magnification.
Discrepant Analysis
Results that were discrepant between the m-RT-PCR-ELAHA and the DFA/CA-DFA algorithm were resolved by discrepant analysis using monospecific PCR assays for each virus. Adenovirus-positive specimens were confirmed using an ADV nested PCR assay targeting a sequence of the hexon gene33
2050-bp upstream from the m-RT-PCR target for this virus. Specimens positive for Flu A were confirmed using a LightCycler (Roche) assay, which targeted the matrix gene.34
The single specimen positive for PIV1 was confirmed by a monospecific RT-PCR assay using primers, which targeted the hemagglutinin neuraminidase gene 161-bp upstream from the m-RT-PCR target for this virus.7
The PIV2 specimen was confirmed using an RT-PCR assay targeting a sequence of the gene coding for the nucleocapsid protein, 46-bp upstream from the sense primer of the multiplex target.35
The additional specimens positive for PIV3 were confirmed using a monospecific RT-PCR assay, targeting the hemagglutinin-neuraminidase gene (unpublished data). RSV-positive specimens were confirmed using a PCR-ELAHA assay that targets the RSV nucleocapsid gene.35
Previous evaluations of these confirmatory tests in our laboratory have shown that each assay is highly suitable for the detection of its respective target (unpublished data).
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Results |
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). The m-RT-PCR-ELAHA detected 23 additional positive specimens (see Table 3
).
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. All influenza B-positive specimens were detected by DFA, but considering these comprised only two specimens, the validity of this result was difficult to interpret. Comparing the conventional methods to m-RT-PCR shows a sensitivity of 79.7% for the DFA and a sensitivity of 88.6% for the DFA/CD-DFA testing algorithm. Of the 396 specimens that were negative by all assays, only five were found to be negative for the internal control sequence (human ERV). All specimens that were positive by m-RT-PCR-ELAHA for respiratory viruses were positive for human ERV. The analytical sensitivity of the m-RT-PCR-ELAHA was greatest for RSV with a value of 5 TCID50 per milliliter of culture. Similarly, the assay detected 10 TCID50 per milliliter of ADV, Flu A and PIV1, 15 TCID50 per milliliter of Flu B and PIV3, with the detection of PIV2 the least sensitive at 40 TCID50 per milliliter.
The specificity of all three methods was found to be 100%. The 23 specimens that were positive by m-RT-PCR-ELAHA alone were also positive by conventional PCR assays targeting different regions of each viral genome. The specificity of the m-RT-PCR-ELAHA was further confirmed by testing RNA and DNA that had been extracted from 41 unrelated viruses, bacteria, yeast, and fungi. No positive reactions were observed. In addition, the specificity of the ELAHA probes was established by testing for cross-reactions of amplification products obtained from the seven target respiratory viruses with unrelated probes. We found there were no cross-reactions in the ELAHA detection step.
The turnaround time for conventional virus detection methods ranged from approximately 2 to 3 hours for DFA to 2 to 3 days for CA-DFA and up to 1 to 2 weeks for virus isolation. The m-RT-PCR-ELAHA assay, on the other hand, had a turnaround time for results that was less than 5 hours. The cost of reagents alone for DFA and CA-DFA in our laboratory was calculated as $USD 16.23, and $USD 19.92, respectively, whereas the m-RT-PCR-ELAHA was $USD 11.07.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An inherent limitation in designing a multiplex PCR assay is the loss of sensitivity that results when combining a large number of primer sets in a single reaction. In this study, we carefully optimized the reaction conditions to obtain maximum sensitivity (data not shown). As a result, despite the presence of eight primer sets in the PCR reaction mixture, the m-RT-PCR-ELAHA was able to specifically detect all virus types tested at a high level of sensitivity. This sensitivity was enhanced by the incorporation of the ELAHA detection step, which was approximately 100-fold more sensitive than conventional agarose gel detection methods.26 As a result, our findings show that PCR assays were more sensitive than traditional diagnostic methods, which is consistent with previous reports.7, 12, 13, 16
Our results illustrated the limitations of virus culture and DFA-based techniques for the detection of viruses in clinical specimens.8, 12, 14, 36 The failure to detect virus by these techniques (DFA/CA-DFA) might be due to low viral load of or the presence of non-infectious virions in the specimen, which resulted from insufficient specimen or inappropriate transport or storage conditions (ie, specimens were kept for extended period at room temperature or higher).13 RT-PCR technology is not affected by these limitations because it is dependent on the presence of viral nucleic acid rather than infectious or intact virions.18
The incorporation of an internal control in a PCR reaction is highly desirable, and we included human ERV to monitor inhibition of the PCR. However, our results showed that the majority of respiratory specimens do not contain inhibitory substances. This low level of inhibition may be attributed to the fact that NPA specimens were placed in 2 ml of VTM, which may have diluted the inhibitors and limited their effect on the PCR. Alternatively, it may reflect the efficiency of the extraction procedure to remove inhibitors that are commonly present in respiratory secretions. Whatever the case, the low incidence of inhibitory substances in our specimens suggests that it may not be cost-effective to incorporate the internal control into the assay. On the other hand, the addition of the internal control would be beneficial for laboratories that use other extraction protocols and experience higher levels of inhibition in their specimens.
The specificity of the m-RT-PCR-ELAHA, the DFA and the CA-DFA assays was 100%. The specificity of the m-RT-PCR-ELAHA was enhanced by the use of the ELAHA detection method, which used oligonucleotide probes to specifically detect the amplification product for each virus. This meant that both the amplification and the detection steps in this assay were virus specific. This gave a greater degree of confidence in the results when compared to conventional amplicon detection by gel electrophoresis, where non-specific or spurious bands may affect result interpretation.26 Also, the ELAHA detection method is a safer alternative to agarose gel detection that uses ethidium bromide, a reported mutagen and suspected carcinogenic agent.26 The specificity of the m-RT-PCR-ELAHA was further demonstrated by the lack of cross-reactions in both the RT-PCR and ELAHA steps when testing RNA or DNA from unrelated viruses, bacteria, yeast, or fungi.
In addition to improved sensitivity and high specificity, we found that the m-RT-PCR-ELAHA offered other significant advantages, including improved result turnaround times and greater cost-effectiveness. However, an experienced operator can perform the DFA faster (3 hours) that the m-RT-PCR-ELAHA (5 hours), but our results showed that testing by DFA alone would result in failure to detect the respiratory pathogen in approximately 20% of PCR-positive specimens. Thus, as an alternative, the m-RT-PCR-ELAHA offers increased sensitivity with a same day turnaround time for results, and in laboratories where DFA might remain the test of first choice, m-RT-PCR ELAHA could be used as a substitute for cell culture-based assays, resulting in improved clinical service.
Our evaluation showed that the m-RT-PCR-ELAHA was more cost-effective than DFA and the CA-DFA. Considering reagent costs alone, the cost per test of the m-RT-PCR-ELAHA was $USD 5.16 to $USD 7.37 cheaper than DFA and the CA-DFA respectively. In addition, labor costs were less for the multiplex PCR, as the training of personnel in this technology and result interpretation were relatively generic, whereas performance and interpretation of cell culture and DFA-based assays is more labor intensive and requires special training. Therefore, to our knowledge, this assay is the only one described thus far that combines maximum sensitivity with high specificity and cost-effective use of reagents and operator time.
Clinically, the rapid and accurate diagnosis of respiratory viruses is important to improve patient management and direct therapy following a specific diagnosis. Identification of the pathogen will limit unnecessary antibiotic usage, and prevent nosocomial spread of viruses such as RSV to hospitalized infants and immunocompromised patients.3, 5, 37 In addition, the m-RT-PCR-ELAHA may be used as a tool to evaluate the effectiveness of, or to justify the development of, a new viral vaccine. In this regard, we suggest that m-RT-PCR ELAHA described here is an effective clinical laboratory technique for the detection of seven common respiratory viruses in samples from patients with respiratory infections.
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Acknowledgments |
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Footnotes |
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Supported by Royal Children’s Hospital Foundation grants RA921–006 and I922–034 which were sponsored by the Woolworth’s "Care for Kids" campaign.
Accepted for publication February 10, 2004.
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