Chinese and US scientists found new methods for virus detection

Two medRxiv articles on the same day: A new method to detect viruses has been found by Chinese as well as other scientists, which can be tested on-site, with results within 45 minutes.

China coronavirus test.
China coronavirus test.




๐Ÿ”น These methods are of great significance for the community and the general population to detect infection with the virus.

The new coronavirus (SARS-CoV-2 or 2019-nCoV) threatens global health, and although it is very similar to SARS-CoV, it is more contagious.

Existing RT-qPCR detection methods often require expensive equipment and trained professionals. Under these circumstances, it is urgent to develop a technique that is fast, accurate, and capable of detecting viruses in the field.

On February 25, preprint website medRxiv issued respectively by the Institute of Applied Biology, Shenyang University of Chemical Technology, Dr. Yin Xiushan team, and the American Royal Oak, Michigan Beaumont Michael B Chancellor team.

New coronavirus test.
New coronavirus test.


The study reports brought by Chinese and foreign scientists revealed reverse transcriptase Loop-mediated isothermal amplification (RT-LAMP) technology had explored new methods for rapid virus detection. 

These two methods are not only simple to operate and can be used in the field, and the detection results can be obtained in 45 minutes.

RT-LAMP is a technology that combines loop-mediated isothermal amplification (LAMP) and reverses transcription to detect RNA directly. When it is coupled with the pH indicator in the reaction mixture, people can know by the color change Test results.

๐Ÿ”น iLACO detects as few as 10 copies of the ORF1ab gene.

Dr. Yin Xiushan's team developed and validated an optimized isothermal LAMP-based detection method, iLACO (isothermal LAMP based method for COVID-19).

In the study, the researchers prepared multiple reactants, which contained a gene dilution (from 1,000,000 to 10 copies) of the synthetic ORF1ab gene sequence.

๐Ÿ”น They found that iLACO was very sensitive and could detect as few as ten copies of the ORF1ab gene.

iLACO.
iLACO.



Finally, researchers used iLACO to evaluate 43 samples that were newly diagnosed by RT-qPCR during the outbreak in Shenyang in 2020.

The results showed that 97.6% (42/43) of the samples verified by qRT-PCR were incubated with 2 ยตl samples for 40 minutes A consistent signal was shown, and only one sample remained unchanged in color after 50 minutes, suggesting that low doses of the virus would cause false negative or spontaneous negative signals.

At present, most RT-qPCR tests use 5ยตl sample input, so the researchers tested whether increasing the sample size would help the test, and found that the test results were inconsistent at this time.

In a second report brought by Michael B Chancellor's team: The researchers used the COVID-19 PCR standard designed from nucleotides 2941-3420 of the COVID-19 Wuhan-Hu-1 complete genome (MN908947). Several primers, temperature range (57-65 ° C) and incubation time (15-45 minutes) were tested to determine the optimal conditions for RT-LAMP. 

๐Ÿ”น Incubate for 30 minutes at 63 ° C for best amplification results.

Researchers performed system-specific tests on the COVID-19, MERS, BtCoV, and MHV viruses and found that the system only responded to samples of COVID-19, using human serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs. 

๐Ÿ”น All children can successfully complete RT-LAMP.

In addition, the researchers sequenced the SARS and flu-related coronavirus sequences of the RT-LAMP primers, and found that the coronavirus primers and a 27-54% presence of nucleotide mismatch, indicating that they cannot detect positive RT-LAMP results.

Considering the possible mutation of the virus, the researchers also checked to see if the RT-LAMP primers matched 27 different COVID-19 isolates from other locations, and found that the degree of mismatch was 0.


COVID-19 detection.

Although this study has some limitations, such as the COVID-19 biosafety level, and because it is not possible to directly detect related viruses, nucleotide oligonucleotides from the same region of these viruses were used, but these experiments did prove the feasibility of the method. 

๐Ÿ”น The specificity of COVID-19 are of great value for screening and virus detection of potential infected persons.

๐Ÿ”น The two studies aimed to find faster, more accurate, and easier-to-use virus detection methods than RT-qPCR detection.