Rapid Identification of Measles Virus Vaccine Genotype by Real-Time PCR

Abstract

During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale.

Keywords: PCR; genotyping; measles; measles vaccine; molecular methods.

FIG 1

Amplification curve from the MeVA RT-qPCR on the Roche LightCycler 480 system. The bundle of the flat curves includes 33 wild-type measles virus specimens comprising the following genotypes: B2, B3, C1, C2, D2, D3, D4, D5, D6, D7, D8, D9, D10, G1, G2, E, H1, and H2. The amplification curves are from MeV vaccine RNA from 10² to 10⁵ copy numbers, assessed in duplicate. The QuantiTect Probe RT-PCR kit was used for these reactions.

FIG 2

Correlation between Cp values of 50 genotype A measles virus specimens tested by MeVA RT-qPCR and the standard MeV RT-qPCR method. The regression line has a slope of 0.88 (0.82 to 0.94, 95% CI), a y intercept of 4.1 (2.2 to 6.0, 95% CI) and an R² value of 0.949 (P < 0.0001). The QuantiTect Probe RT-PCR kit was used for these reactions.

FIG 3

Negative effect of the use of the SuperScript III Platinum One-Step quantitative RT-PCR kit on the specificity of the MeVA RT-qPCR for the vaccine genotype. (A) The use of SuperScript III on the Roche LightCycler 480 platform caused a significant rise in the baseline of the amplification curves for genotype D10, D8, and B3. (B) Results of the use of SuperScript III on an ABI 7500 platform in amplification curves from wild-type measles virus RNA.

FIG 4

Alignment of the N gene region (positions 478 to 548) amplified by the MeVA RT-qPCR. The alignment includes examples of each genotype available on GenBank for this region, except for genotype D9, which was sequenced from one of our archival specimens. Row 1, 31 identical sequences from various vaccine strains; row 2, MVi/Maryland.USA/54 (A); row 3, two vaccine strains showing a 1-nt difference in the forward primer region; row 4, MVi/Yaounde.CMR/12.83 (B1); row 5, MVi/Libreville.GAB/84 (B2); row 6, MVi/Ibadan.NIE/971 (B3); row 7, MVi/New_York.USA/94 (B3); row 8, MVi/Maryland.USA/77 (C2); row 9, MVi/Illinois.USA/89/1 (D3); row 10, MVi/Montreal.CAN/89 (D4); row 11, Bankok.THA/12.93 (D5); row 12, MVi/New_Jersey.USA/94/1 (D6); row 13, MVs/Dundee.UNK/82 (D7); row 14, MVi/BritishColumbia.CAN/13.10/1 (D8); row 15, MVi/Manchester.GBR/30.94 (D8); row 16, MVs/Ontario.CAN/14.14 (D9); row 17, MVi/Berkeley.USA/83 (G1); row 18, MVi/Amsterdam.NLD/49.97 (G2); row 19, MVi/Gresik.IDN/17.02 (G3); row 20, MVi/Hunan.CHN/93/7 (H1); row 21, MVi/Beijing.CHN/94/1 (H2).

FIG 5

Specificity of the MeVA RT-qPCR assay, using an Applied Biosystems 7500 platform. Synthetic RNA from the six active wild-type measles virus genotypes (B3, D4, D8, D9, G3, and H1) was tested. Panel A shows detection of 10⁷ copies of RNA/reaction in the MeV qPCR assay, and panel B shows the lack of amplification of 10⁷ copies of RNA/reaction in the MeVA RT-qPCR assay. The QuantiTect Probe RT-PCR kit was used for these reactions.