Mmmm...
A "novel virus", heh?
This was published on July 1, 2006. The emphasis is mine.
Preparation of a Chimeric Armored RNA as a Versatile Calibrator for Multiple Virus Assays
Preparation of a Chimeric Armored RNA as a Versatile Calibrator for Multiple Virus Assays
Associated Data
To the Editor:
As with all diagnostic techniques, molecular testing requires careful quality control (1)(2)(3). In detection of RNA viruses, which are often present at low concentrations and are prone to degradation, stringent monitoring is needed for all aspects of assay performance, including virus lysis, RNA isolation, reverse transcription, amplification, and detection steps. Among many proposed RNA control preparations (4)(5), armored RNA is currently the most suitable for clinical applications as it carries the viral RNA target of interest in a form that is ribonuclease-resistant, noninfectious, and stable after prolonged incubation in clinical matrices, and the preparations are substantially less expensive to manufacture than virusinfected plasma (6)(7)(8). Thus, armored RNA has been applied as a positive control for a variety of RNA viruses (9).
Because most commercial armored RNA preparations contain exogenous sequences of <500 nucleotides (9), separate armored RNA species are often prepared for calibration of each target in multiple virus assays. To reduce costs and simplify multivirus detection, we are seeking to produce a single chimeric armored RNA species that might be used as a positive control for multiple viral targets. We consider this task to be feasible because the inventors of armored RNA predicted that, theoretically, at least 2 kb of nonbacteriophage RNA sequence might be encapsulated (8). As proof of this principle, we tried to directly package a 1200-nucleotide–long foreign RNA sequence containing gene fragments of hepatitis C virus (HCV), HIV-1, severe acute respiratory syndrome coronavirus 1 (SARS-CoV1), and SARS-CoV2 into the original armored RNA production vector pAR-1 (8).
We spliced the 4 target cDNA sequences by overlapping extension (10). After cloning the 4-target chimeric sequence (see the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue7/ for the sequence information of the 4 fragments as well as the primers and probes used) into pAR-1, we used a simple but straightforward procedure to confirm the production of armored RNA and to purify it. Briefly, after induction of armored RNA production, we treated the supernatant of Escherichia coli cell lysate with RNase A and DNase I. On testing with agarose gel electrophoresis, if armored RNA was produced, a single DNA band of ∼1.5 kb might be visible. We then cut the band from the gel and put in a dialysis bag for electroelution. Using this method, we successfully expressed and purified the chimeric armored RNA. We used a pure RNA transcript fragment of SARS-CoV2 (BNI) to calibrate the chimeric armored RNA, then used the chimeric armored RNA to prepare calibrators of the 4 real-time reverse transcription-PCR (RT-PCR) assays (Fig. 11 ; also see Fig. 1 in the online Data Supplement) based on displacing probes (11). The linear range for each assay did not change when the calibrators were stored at 37 °C for 2 weeks, at 4 °C for 6 months, or at −20 °C for 1 year.
Calibration of the real-time RT-PCR assay for HCV, HIV-1, SARS-CoV1, and SARS-CoV2.
We diluted purified and calibrated armored RNA with pooled normal human plasma supplemented with 1 g/L sodium azide and prepared 200-μL aliquots by 10-fold serial dilution to obtain samples containing 1010 to 101 copies. From these materials, we isolated template RNA ranging from 1010 to 101 copies (from left to right) for RT-PCR assays. Water was used as a negative control. All RNA templates were assayed in a single run using a diagnostic reagent set (Intec) for each individual virus. Real-time RT-PCR was conducted on an iCycler iQ thermal cycler (Bio-Rad).
Our work indicates that multiple target sequences can be encapsulated into a single armored RNA species to serve as a common calibrator for detection of different RNA viruses. Chimeric armored RNA of even larger size may be prepared similarly, as indicated by our finding that by deleting some disposable sequences between the multiple cloning site and the transcription terminator, we were able to increase packaging capacity of the pAR-1 vector without affecting packaging efficiency (data not shown). Thus, the chimeric, multitarget approach for armored RNA preparation is practical and could reduce the labor and cost for quality control of multiplex RNA virus assays.
Acknowledgments
We thank Xilin Zhao and Karl Drlica for critical comments on the manuscript. This work was partially supported by the Natural Science Foundation of Fujian Government (2003Y004), by the Xiamen Municipal Commission of Science and Technology Key Program, and by the Xiamen University Action Project.
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