Comparison of Four Methods Using Throat Swabs To Confirm Rubella Virus Infection

Abstract

Laboratory tests are essential for confirming sporadic cases and outbreaks of rubella. Detection of rubella virus is often necessary to confirm rubella cases and to identify specimens to be used to characterize wild-type rubella viruses. The sensitivities of four methods for detecting rubella virus infection using throat swabs, which had been collected in Henan and Anhui provinces in China, were evaluated. The methods used were reverse transcription (RT)-PCR followed by Southern hybridization using RNA extracted directly from clinical specimens, virus growth in tissue culture followed by virus detection by RT-PCR, low-background immunofluorescence in infected tissue culture cells using monoclonal antibodies to the structural proteins of rubella virus, and a replicon-based method of detecting infectious virus. Among these four methods, direct RT-PCR followed by hybridization was the most sensitive method; the replicon-based method was the least difficult to perform.

Rubella Virus Genotypes in the People's Republic of China between 1979 and 2007: a Shift in Endemic Viruses during the 2001 Rubella Epidemic

“…The viral RNA was extracted from infected cells after additional passages by using the QIAamp viral RNA extraction minikit (Qiagen, Valencia, CA). The presence of viral RNA was detected via reverse transcription-PCR (RT-PCR), which amplified a 185-nt fragment of the E1 coding region as previously described (36).…”

“…Specimens were inoculated onto monolayers of African green monkey kidney (Vero) cells or Vero/SLAM cells, according to standard methods (36). Cells inoculated with clinical specimens were incubated at 35°C for 7 days.…”

“…To control rubella epidemics and prevent CRS, routine rubella vaccination was instituted in the Chinese national immunization program in 2007. Two types of rubella vaccine are currently available in China (36). The BRDII attenuated vaccine strain of rubella virus, derived from a wild-type rubella virus isolated from a patient during an epidemic in Beijing in 1979 and attenuated through 30 lowtemperature passages on a diploid cell line (23), was introduced in 1994 in some parts of China.…”

Confirmation of Rubella within 4 Days of Rash Onset: Comparison of Rubella Virus RNA Detection in Oral Fluid with Immunoglobulin M Detection in Serum or Oral Fluid

“…1). RT-PCR for the generation of a 185-bp amplicon was performed with a SuperScript OneStep RT-PCR kit (Invitrogen, Carlsbad, CA), as described previously (25), except that the number of amplification cycles was increased from 35 to 40. In addition, for many of the OF-derived RNAs (the first 45 samples processed and then 1 sample in every batch of 10), amplification of a 150-bp region of the ␤-actin gene was performed as a control for the quality of the RNA extractions.…”

“…The virus titer in throat swabs, however, usually reaches a peak titer on the day of rash onset and the titers in throat swabs decline more slowly than those in blood, so that virus can be detected for up to 5 to 7 days after rash onset (6). Several RT-PCR assays for the detection of the RV genome in clinical samples have been described (3,7,15,16,20,25). Templates for the determination of viral sequences for molecular epidemiology can also be made by using RT-PCR.…”