New cases of Coronavirus nCoV-2019 are being confirmed every day, in China and now, around the world. There are thousands of cases (roughly 4,500) confirmed, most of which are in the epicenter of China, but include (at the time of the writing of this article) 15 other countries.

cdc.gov
Locations with confirmed 2019-nCoV cases including China, Hong Kong, Macau, Taiwan, Australia, Cambodia, Canada, France, Japan, Malaysia, Nepal, Singapore, Thailand, The Republic of Korea, United States, Vietnam [cdc.gov]
In the United States, there are currently five confirmed cases, with over 100 more currently under investigation. The CDC is conducting screenings at 20 American airports that receive 90% of all passengers from China.

How are cases of nCoV-2019 identified? Especially given the confounding factor that coronavirus causes a respiratory infection—with symptoms such as runny nose, headache, dry cough, sore throat, and fever—and certain areas of the world are in the middle of flu season.

Using an old trick for a new outbreak

Coronaviruses have genomes encoded by RNA. Therefore, a standard PCR cannot be used to detect the presence of the virus. However, with the small addition of a reverse transcriptase step (which converts RNA to DNA) the viral genome can be detected. Indeed, this technique that has been around for decades, called RT-PCR, now lies squarely in the middle of the outbreak.

Up until now, all coronavirus testing in the United States has been done at the CDC. But, Nancy Messonnier, MD, director, National Center for Immunization and Respiratory Diseases (NCIRD) noted in a press briefing on Monday that the CDC is making detection kits and is “working on a plan” so that “priority states get these kits as soon as possible.”

In China, where the number of cases is much larger and testing is in high demand, testing kits have been hard to come by. Reuters reported on January 27th that “testing kits for the disease were not distributed to some of Wuhan’s hospitals until about January 20.”

On January 26th, China’s regulatory agency, the National Medical Products Administration (NMPA) announced the approval of four new coronavirus detection products.

Included in those are real-time fluorescent RT-PCR kits made by the Chinese genome sequencing company BGI. Not only has BGI donated the first 10,000 kits to support the response in Wuhan, and sent two of their DNBSEQ-T7 sequencers as well, the chairman and co-founder Wang Jian and a team of employees arrived in Wuhan on January 25th to lead BGI’s emergency response.

According to the company’s website, they set up “an emergency command center in Wuhan to mobilize efforts and expand the manufacturing capacity, with staff working around the clock to produce the testing kits and arrange storage, transportation, and other logistics.” BGI has now released a total of 40,000 test kits to hospitals and disease control centers around China. They noted that they can produce about 50,000 kits a day and have about 100,000 in stock.

The Chinese company Liferiver Biotech took just over 10 days to develop their coronavirus detection kit, according to reporting by CGTN, the official website for China Global Television Network. They noted that, since the genomic sequence of nCoV-2019 was published on January 10th, employees at the company have maintained an around the clock schedule to develop the kit that eliminates SARS 2003 and Bat SARS-like virus strains, making the detection of nCoV-2019 virus specific. Liferiver asserts that their kit can detect the presence of the virus in less than two hours, with 100% accuracy, and that their production capacity can test 200,000 people a day.

Hong Li, PhD, department head of reagent services, R&D department, at GenScript—a company with headquarters in both China and the United States—told GEN that their kit tests for the presence of four multiple nCoV-2019 genes: the O, R, N, and E gene. Typical of RNA viruses, coronaviruses mutate frequently. Detecting multiple genes simultaneously reduces the risk of missing detection that could occur with genomic variation. In addition, Li noted that they can detect the virus at a very early stage.

Eric Wang, VP of marketing at GenScript, explained to GEN that this kit is currently for research processes and is not designed for hospital use. But because of the shortage of the detection methods in China, Wang noted that they are currently seeking approval for clinical use which they expect will take weeks. In the meantime, the kit can be used for research purposes.

The Genscript detection kits, similar to other kits, consist of primers, reverse transcriptase enzyme, buffer, and the dye for PCR machine calibration. The first step in the process is to isolate RNA from the patient (blood or sputum samples). Once isolated, reverse transcriptase converts the ssRNA to DNA. The cDNA can then be amplified with primers and Taq polymerase to enhance the signal.

In addition to their detection efforts, Genscript noted that they have received urgent requests from partners to synthesize the genes of nCoV-2019 as quickly as possible, so that companies, universities, and government agencies can get to work on the vaccine and therapeutic development.

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