Sensitive detection of SARS coronavirus RNA by a novel asymmetric multiplex nested RT-PCR amplification coupled with oligonucleotide microarray hybridization

Abstract

We have developed a sensitive method for the detection of specific genes simultaneously. First, DNA was amplified by a novel asymmetric multiplex PCR with universal primer(s). Second, the 6-carboxytetramethylrhodamine (TAMRA)-labeled PCR products were hybridized specifically with oligonucleotide microarrays. Finally, matched duplexes were detected by using a laser-induced fluorescence scanner. The usefulness of this method was illustrated by analyzing severe acute respiratory syndrome (SARS) coronavirus RNA. The detection limit was 10(0) copies/microL. The results of the asymmetric multiplex nested reverse transcription-PCR were in agreement with the results of the microarray hybridization; no hybridization signal was lost as happened with applicons from symmetric amplifications. This reliable method can be used to the identification of other microorganisms, screening of genetic diseases, and other applications.

Fig. 1.

Schematic representation of the multiplex PCR used in this work. (a), Targets for amplification. Only two targets are shown here. (b), Gene-specific primer amplifications, which are the same as ordinary PCR except that two different universal sequences irrelevant to the targets were added to the 5′ end of the specific primers. (c), Universal primer amplification. In this reaction, the amplicons of the gene-specific primers served as templates. sf, the forward gene-specific primer; sr, the reverse genespecific primer; uf, the forward universal primer; ur, the reverse universal primer. All primers are added to one tube. (A) For symmetric amplification, uf and ur are used with equal molar amounts. (B) For asymmetric PCR, the uneven primers uf and ur were used, and in some cases uf was even absent from the reactions.

Fig. 2.

The illustration of the microarray designed for detection of SARS CoV. BC, DMSO spotted as blank control; QC, Hex-labeled oligonucleotide used for quality control of surface chemistry; IC, internal control probe for nested RT-PCR and hybridization; EC, external control probe for hybridization; NC, negative control probe for hybridization; 11 and 24, probes selected from SARS CoV’s ORF 1a; 40, probe selected from the ORF of SARS CoV for nucleocapsid protein; 44, probe selected from the ORF of SARS CoV for spike glycoprotein.

Fig. 3.

Quantitative analysis of SARS CoV RNA by means of real-time RT-PCR. (a–d), quantification standards (HPA-CoV LC QS 1–4) supplied within the kit by the manufacturer. Concentrations of SARS virus RNA in (a-d) are 10¹, 10², 10³, and 10⁴ copies/µL, respectively. (s), sample for quantification. Before real-time RT-PCR, the SARS viral RNA for quantification was diluted 10⁵ times.

Fig. 4.

Electrophoretic results of multiplex nested RT-PCR amplicons. (A–D), SARS CoV RNA and human total RNA at concentrations of 10₃, 10₂, 10₁, and 10⁰ copies/µL or 167, 16.7, 1.67, and 0.167 ng/µL as the templates for RT-PCR in lanes 1–12 (three parallel reactions were performed). (a–d), Ratios of universal primers uf to ur were 1:1, 1:25, 1:100, and 0:1, respectively. Lane M, molecular size marker; lane N, negative control, for which the same reaction was performed but without the addition of the virus RNA; lane B1, blank control, in which reaction DEPC-treated H₂O was used as the template. The arrow shows the visible amplicon (257 bp) for the pair of primers 40 in this reaction.

Fig. 5.

Part of the hybridization results. (A–D), SARS CoV RNA and human total RNAat concentrations of 10³, 10², 10¹, and 10⁰ copies/µL or 167, 16.7, 1.67, and 0.167 ng/µL, respectively, were used as templates for RT-PCR. (a–d) In multiplex PCR the ratios of universal primers uf to ur were 1:1, 1:25, 1:100, and 0:1, respectively. In the three parallel multiplex nested RT-PCR reactions, only one hybridization image (lane 3, 6, 9, and 12 in Fig. 4) was shown. NC, negative control; BC, blank control. All NC (BC) had the same hybridization images, so only one is shown here. For probe 40, no hybridization signal was observed in (a), but signal was seen in (b–d).