A two-tube multiplex reverse transcription PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes

Abstract

There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20-200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China.

Figure 1

Specificity analyses of the the two-tube assay based on the automatic electrophoresis. All positive controls were identified successfully, and no mispriming was observed in either tube. Lanes 1 to 9 show the results of amplification of HRV (148 bp), FluB (171 bp), CoV 229E (189 bp), CoV OC43 (208 bp), CoV HKU1 (226 bp), FluA (sH1N1) (257 bp and 276 bp), FluA (H3N2) (276 bp), PIV1 (286 bp) and Adv (342 bp), respectively. Lanes 11–17 show the results of amplification of RSVA (163 bp), CoV NL63 (179 b), PIV2 (198 bp), HMPV (212 bp), PIV3 (233 bp), RSVB (280 bp), and HBoV (298 bp), respectively. For the viral targets that used clinical specimens as positive controls, including HBoV, CoV HKU1, and CoV NL63, a single PCR product was detected in addition to the internal control peak (126 bp). Lanes 10 and 18 are the negative controls (distilled water) of tube 1 and tube 2, respectively. Lane MM, molecular marker.

Figure 2

Sensitivity analyses of the two-tube assay based on the automatic electrophoresis. Lanes 1 to 5 contain PCR products of pre-mixed viral targets in tube 1 of 2  ×  10⁶ to 200 copies. Lanes 7 to 11 contain PCR products of pre-mixed viral targets in tube 2 of 2  ×  10⁵ to 20 copies. Lanes 6 and 12 are the negative controls of tube 1 and tube 2, respectively. Lane MM, molecular marker. The detection sensitivity in tube 1 with 9 pre-mixed viral targets was 2000 copies per reaction and 200 copies per reaction in tube 2 with 8 pre-mixed templates. Only the amplicons of sH1N1 and CoV 229E were absent with dilutions of 200 copies of pre-mixed viral targets in tube 1 (Lanes 5), and only the amplicons of CoV NL63, HMPV, and RSVB were absent with dilutions of 20 copies of pre-mixed templates in tube 2 (Lanes 11). The primer dimers which were inevitable in the multiplex PCR are increasingly apparent with the dilution of template.