Banna Virus Detected by Reverse Transcription-Loop-Mediated Isothermal Amplification

BAV is considered to be an emerging pathogen that can result in human infections with possible manifestation of fever and viral encephalitis.

Reverse transcription-loop mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification approach that amplifies reverse transcribed DNA from RNA using strand displacement DNA polymerase under isothermal conditions. Due to its rapidness, simplicity, sensitivity and specificity, RT-LAMP has been successfully applied in the detection of various RNA viruses.

Scientists at the Wuhan Institute of Virology (Wuhan, China) designed a set of six specific primers to target the segment 12 of BAV, and the reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) method. The team used various cells and spiked samples to test the RT-LAMP method.

In running the RT-LAMP assay, a DEAOU RNA Amplification Kit (RT-LAMP) was used. One step RT-PCR amplification for BAV was performed using Prime Script One Step RT-PCR Kit Ver.2. RNA was extracted from 140 μL of BAV-infected C6/36 cell culture supernatant, BAV-spiked human serum or filtered mosquito homogenate samples using the QIAamp Viral RNA Mini Kit.

The team reported that the amplification of the RT-LAMP assay can be obtained within 40 minutes at 65 °C. The results from specificity showed that only target BAVs RNA including genotypes A, B and C were amplified and the assay demonstrated a sensitivity of 3.6 × 10−2 PFU/mL, which was higher than conventional RT-PCR measurement. A good reliability for the assay was presented in the further evaluation for BAVs RNA from serial diluted BAV-spiked serum and 47 pools of field mosquito samples.

The authors concluded that they had successfully developed a RT-LAMP assay for the detection of BAV, which provides a potential new molecular diagnostic test for BAV that could be applied in the field or clinic in the future, and that may contribute to the preparedness for future outbreaks of a BAV endemic, especially for regions with limited resources available. The study was published online on November 2, 2018, in the International Journal of Infectious Diseases.

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Wuhan Institute of Virology