Since the outbreak of novel coronavirus pneumonia COVID-19, as of March 1, more than 80,000 people have been diagnosed and nearly 3,000 deaths have occurred worldwide, but there are still many clinically suspected cases that cannot be diagnosed. A joint team from Wuhan University has developed an innovative nanopore-targeted sequencing assay that can significantly increase the detection rate of positive viruses and enable same-day simultaneous detection and monitoring of viral mutations in 10 major categories of 40 common respiratory viruses, including Neovirus.
Previously, the diagnosis of NIV relied on qPCR nucleic acid testing, but this method showed a high false-negative rate and low sensitivity, with a positive detection rate of only 30% to 50%, resulting in "false-negative" testing in patients with high clinical suspicion of NIV infection or in "pseudo-cured patients" with low viral latency. The phenomenon of "false negatives" is frequent. In addition, qPCR nucleic acid testing cannot simultaneously detect other respiratory viruses that are highly prevalent in the fall and winter and have symptoms similar to those of NCC, making outbreak prevention and control and patient triage management extremely challenging.
Experts and scholars who are in the front line of the epidemic area look at the situation and are in a hurry. Prof. Tiangang Liu from Wuhan University School of Pharmacy, Prof. Yan Li and Prof. Li-Li-Ra Yu from Wuhan University People's Hospital, and Dr. Aisi Fu, General Manager of Wuhan Zhenxi Medical Laboratory Co., Ltd. quickly formed a joint team to develop an innovative Nanopore Targeted Sequencing (NTS) assay, which combines targeted amplification of viruses with long read length, high performance, and low cost. NTS combines the advantages of targeted amplification of viruses and nanopore sequencing with long read length and real-time data output to achieve the first highly sensitive and accurate simultaneous detection of SARS-CoV-2 and other 10 major classes and 40 respiratory viruses within 4 hours after sequencing, and its minimum detection limit is 100 times higher than that of the currently widely used qPCR. The method also enables the detection of novel coronavirus genomic variation and monitoring of virus variation-induced changes in virulence and transmissibility.
On March 3, the team will publish a paper entitled Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses ("Nanopore target sequencing for accurate and comprehensive detection of new coronaviruses") on the preprint platform medRxiv. Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses").
The team used NTS for detection of NIV in Wuhan hospital.
The NTS technology developed by the team is unique in that it is not limited to the loci currently recommended by the Chinese or US Centers for Disease Control (CDC) in qPCR methods, but extends the detection range to 9 genes, 12 loci, and nearly 10 kb regions, comprehensively covering the major genetic regions of the viral genome and 100% of the virulence-related important genes in the viral genome, thus significantly increasing the detection range of the viral genome by 100 times. The detection range of viral genome is increased by 100 times, thus significantly improving the sensitivity and accuracy of detection. In contrast, the qPCR method only targets 2-3 loci on the viral genome, covering <0.5% of the viral genome, and slight deviations in the sampling, storage and testing process can lead to lower efficiency of the PCR detection method targeting only a few loci, or even missed detection, resulting in "false negatives", and the detection If the region is mutated, the test results will be invalidated.
Comparison of NTS and qPCR methods
Liu Tiangang introduced, qPCR method is like using a sniper rifle to hit the virus nucleic acid in the sample, there is a certain probability of missing, while NTS technology is not using a sniper rifle, but casting a dozen nets at the same time, so that the probability of capturing the virus nucleic acid is greatly increased, not only that, but also the sequence can be read out at the same time of capture. In this way, not only can a single test confirm the diagnosis of a new crown, but other common respiratory viruses, including bocavirus, rhinovirus, human plasmopneumovirus, respiratory syncytial virus, coronavirus, adenovirus, parainfluenza virus, influenza A virus, influenza B virus, and influenza C virus, can also be detected simultaneously, providing a definitive basis for physician triage. More importantly, it can also detect whether mutations in genes associated with virulence occur during virus transmission, which can rapidly inform subsequent epidemiological analysis.
In the study, the team tested both NTS and qPCR methods in parallel. The results showed that of 45 samples from patients with highly clinical suspicion of new crown infection, NTS identified a total of 34 positive samples, 15 more than qPCR; of 16 patients with clinically confirmed new crown infection, all tested positive for NTS and only 9 positive for qPCR. The clinical confirmed samples showed a 43.8% increase in positive detection rate for NTS over qPCR. In addition, for high concentration viral samples, NTS can detect positives in only 10 minutes of sequencing, and even for very low concentration viral samples, it only takes 4 hours of sequencing to complete the detection, and the whole process from sample receipt to result is controlled in 6-10 hours. levels of hospital use.
Parallel testing of 16 samples with confirmed NIV infection
At present, the high sensitivity, accuracy, speed and ease of operation of NTS are of great significance in confirming suspected cases as soon as possible and helping to prevent and control the epidemic!
