Objective: To develop the modified molecular beacon-based dual fluorescent PCR assays for detection of SARS virus. The assay was applied to the early clinic diagnosis & animal tracking.
Methods: On the basis of the obtained core sequence of open reading frame 1b of the coronavirus polymerase gene sequences, which was published in GenBank, using modified molecular beacon probe, artifical virus techinique and two different fragments amplification with different fluoresce, one set of primers and probe were designed. Then fluorescent PCR assays for specific and sensitive detection of the SARS virus was established, while the ELISA & the traditional method were used as control. 368 clinical specimens such as the throat swab, serum, feces, and urine from different cases, 52 cell cultures and 50 animal specimens were detected by the molecular beacon-based PCR.
Results: The sensitivity of real -time PCR was 10 - 100 copies/ml, there was no cross reaction to other respiratory viruses such as influenza virus etc. Of 368 specimens, 20 were positive by using molecular beacon-based fluorescence PCR. The positive rate of SARS case (10/47) were 21.27%, the positive rate of the throat swab of SARS cases (10/23) were 43.87% . Among 52 SARS cell cultures, 29 were positive. The positive rate of SARS cell cultures was 55.77% . Of 50 animal specimens, 23 were positive. The positive rate was 46%. Furthermore, SARS virus RNA was detected in feces and in serum during the acute phase.
Conclusion: The molecular beacon-based PCR is sensitive and specific, it could be applied to the early diagnosis and animal tracking. This molecular beacon-based PCR kit is useful for the different units.