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Published on: May 2, 2019
The use of oral fluid samples spotted on filter paper for the detection of measles virus using nested rt-PCR
Sogol Sheikhakbari1, Talat Mokhtari-Azad, Vahid Salimi
1School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Abstract:
Measles is the leading cause of death in infants, although a vaccine is available for its prevention. At this stage of measles elimination and eradication, it is so important to confirm clinically diagnosed measles cases in the laboratory but, developing countries have troubles in collecting and maintaining the cold chain of the specimens while transporting them to the laboratories. Therefore, filter papers are good candidates for simplification of specimen collection and transportation. In this research, the effects of the temperature, at which the dried specimens were kept, and the time duration the dried specimens were kept before being tested, were studied. Since there were not enough patients' oral fluid samples available, a nested reverse transcriptase PCR (RT-PCR) that detected measles virus (MV) from dried filter papers was set up using MV infected cells diluted in sterile phosphate-buffered saline (PBS). Dried specimens were stored at -25°C, 4°C, and room temperature for 1 day, 1, 2, and 3 weeks before being tested. This method was then applied to filter paper oral fluids collected from nine clinically diagnosed measles patients in Iran in 2010 which were tested after being kept at room temperature for 1 day, 1 and 3 weeks after preparation. The results showed that dried oral fluids on filter papers are reliable specimens for the detection of MV RNA using nested RT-PCR, but the nested RT-PCR results of low titer viruses dried onto filter papers are not reproducible and reliable.
Insights
Dried filter paper specimens reliably detect measles virus RNA using RT-PCR, simplifying collection and transport in developing countries. However, low virus titers on filter paper may yield unreliable results.
Area of Science:
- Virology
- Molecular Diagnostics
- Public Health
Background:
- Measles remains a significant cause of infant mortality globally, despite vaccine availability.
- Laboratory confirmation of measles cases is crucial for elimination efforts, but cold chain maintenance for specimen transport poses challenges in developing nations.
- Filter paper offers a promising solution for simplified specimen collection and transportation.
Purpose of the Study:
- To evaluate the stability of measles virus (MV) RNA on dried filter paper specimens under various storage conditions.
- To assess the reliability of a nested reverse transcriptase PCR (RT-PCR) for detecting MV RNA from filter paper samples.
- To determine the feasibility of using filter paper oral fluids for measles diagnosis in resource-limited settings.
Main Methods:
- A nested RT-PCR assay was developed to detect MV RNA from infected cells dried onto filter paper.
- Dried specimens were stored at -25°C, 4°C, and room temperature for durations of 1 day to 3 weeks.
- The method was applied to filter paper oral fluid samples from clinically diagnosed measles patients in Iran, stored at room temperature for 1 day, 1 week, and 3 weeks.
Main Results:
- Dried oral fluids on filter papers proved to be reliable specimens for detecting measles virus RNA via nested RT-PCR.
- Storage temperature and duration affected the reproducibility of results, particularly for low-titer viruses.
- The nested RT-PCR method demonstrated potential for diagnosing measles from filter paper samples under various storage conditions.
Conclusions:
- Filter paper collection and storage of oral fluids is a viable strategy for measles virus RNA detection using nested RT-PCR.
- While generally reliable, the method's accuracy for low viral loads requires further optimization.
- This approach can significantly aid measles surveillance and diagnosis in resource-limited settings, supporting global elimination goals.
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