Nanopore : Israeli single-molecule method for coronavirus testing reduce by 100-fold the quantity of chemicals needed

Nano team at the Technion says it can check molecules one by one, meaning labs won’t need to spend hours ‘amplifying’ samples to generate more material for analysis

Israeli nanotechnologists say that they have found a way to examine molecules one by one for coronavirus, and eliminate the most time-consuming process in COVID-19 test analysis.

In regular virus testing, lab teams need to massively increase the number of molecules they have from each patient’s sample, through a process known as amplification. That typically takes between one and two hours, and requires special chemicals. Millions of molecules are needed before a sample can be analyzed.

But the method just developed in Haifa, tested, and outlined in the peer-reviewed journal ACS Nano, requires just 100 molecules, eliminating all need for amplification, bio-engineer and nanotechnology expert Amit Meller told The Times of Israel Monday.

“We have developed a way to pass molecules, one by one, through a tiny nano hole,” said Meller, of the Technion-Israel Institute of Technology. “The hole is called a nanopore, and isn’t new, but this is the first time it has been deployed for RNA testing for the coronavirus.”

He said that as well as being used for the coronavirus, his analysis method can be deployed in screening for secondary cancers, and said he hopes to see it quickly commercialized for both uses. He said the key is that it retains a level of precision in analysis of ribonucleic acid, RNA, “which is essential in both contexts we studied – RNA biomarkers of metastatic cancer and the SARS-CoV-2 virus.”

His method starts in exactly the same way as existing tests: The patient is swabbed, their sample is dissolved in liquid, and their ribonucleic acid, or RNA, is converted to DNA, which is suitable for screening.

But instead of amplifying the sample, it is immediately examined with the nanopore, one molecule at a time, and lab workers assess whether a cancer biomarker or SARS-CoV-2 is present, and if so, in what quantity.

Using the nano method, the quantity of chemicals needed is reduced by 100-fold compared to regular lab analysis, according to Meller. “Our advances will potentially cause a significant reduction in the cost of testing,” he said.

Discussing the benefits of eliminating amplification in COVID-19 and cancer screening, he said: “Current lab methods can’t look at individual molecules, so amplification is needed, which doesn’t just take time, but also reduces accuracy.

“If you think of when you try to amplify a very quiet sound using an audio amplifier, you end up getting extra interference and noise, which is exactly what happens when amplifying for coronavirus tests. This is why results aren’t always accurate. Because we are eliminating amplification, we expect our method to boost accuracy.”

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A detection technology, nanopore targeted sequencing (NTS), for the accurate and comprehensive detection of SARS‐CoV‐2 and other respiratory viruses within 6–10 h is developed, which is suitable for the identification of suspected cases and used as a supplementary technique for the SARS‐CoV‐2 test. NTS can also monitor mutations in the virus and the type of virus.

The novel coronavirus disease (COVID‐19) has spread worldwide, resulting in numerous cases of morbidity and death. Generally, COVID‐19 has an incubation period of 2–7 days,[ 1 ] with no obvious symptoms, during which time the virus can spread from infected to uninfected individuals.

Therefore, early accurate diagnosis and isolation of patients is key to controlling the COVID‐19 pandemic. Although antibody‐based detection methods are rapid, they are readily affected by factors, such as sample hemolysis, the presence of fibrin, bacterial contamination, and patient autoantibodies, resulting in a high false positive rate.

Therefore, nucleic acid detection continues to be the gold standard for COVID‐19 diagnosis, with several such methods having been employed for detection of the COVID‐19 causative virus, SARS‐CoV‐2.[ 2 ] Specifically, real‐time reverse transcription‐polymerase chain reaction (RT‐qPCR) is currently the most popular testing method for detecting SARS‐CoV‐2. RT‐qPCR is specific, rapid, and economic; however, it is unable to precisely analyze amplified gene fragment nucleic acid sequences.

Thus, positive SARS‐CoV‐2 infection is confirmed by monitoring one or two sites (depending on manufacturer guidelines). Furthermore, RT‐qPCR exhibits high false‐negative rates in clinical applications,[ 3 ] which can facilitate infection transmission through delayed patient isolation and treatment, resulting in continued COVID‐19 spread.

Several novel intelligent methods for RNA virus detection have been developed, including combining toehold switch sensors,[ 4 ] which can bind to and sense virtually any RNA sequence, using paper‐based cell‐free protein synthesis.

This method has been applied for the detection of Ebola and Zika virus[ 5 , 6 ] and thus should theoretically be capable of rapid and high‐throughput SARS‐CoV‐2 detection as well. Additionally, the SHERLOCK method based on CRISPR/Cas13, can detect Zika, Dengue, and SARS‐CoV‐2 virus.[ 7 , 8 ]

Similarly, the DETECTR method based on CRISPR/Cas12 has been developed for the detection of SARS‐CoV‐2.[ 9 ] Additional new methods based on isothermal PCR amplification are also available, such as Abbott’s ID Now instrument, which can interpret results in minutes.

However, the requirement for specific RNA regions as targets may negatively affect detection rates as mutation of the target region may limit target availability.

Indeed, a 382 nt region of the SARS‐CoV‐2 genome was found to be deleted in Singapore.[ 10 ] Similar deletion events may occur in other regions of the SARS‐CoV‐2 genome, thereby increasing the risk of acquiring false‐negative results if the detection sites are located within the deletion regions.

Sequencing platforms constitute an additional recommended detection method. These platforms are widely applied for pathogen identification and monitoring of virus evolution,[ 11 , 12 ] including that of SARS‐CoV‐2.[ 13 ]

Previous massive parallel sequencing platforms sequence DNA by detecting optical or chemical signals. Sequencing by synthesis used in Illumina (the most widely used massive parallel sequencing platform) requires multiple sequencing cycles, each of which takes several minutes to complete, and analyzes a single base of each DNA fragment.

Hence the sequencing process generally takes 0.5 to 3 days according to the requirement for read length and data output.

Moreover, the sequencing data cannot be applied for further analysis until the entire sequencing process is complete. Nanopore sequencing directly detects changes in currents generated when DNA/RNA molecules pass through a nanopore protein.

The speed of DNA/RNA passing through a nanopore protein is incredibly high (≈450 base s−1 for DNA and 80 base s−1 for RNA).

The electrical signal corresponding to each nucleic acid that passes through the nanopore protein can be recorded in real‐time and used for subsequent sequence analysis immediately.[ 14 ]

The nanopore metagenome method has been shown to effectively detect the respiratory bacterial infection[ 15 ] and virus[ 16 , 17 ] directly from clinical samples. Pathogens and antibiotic resistance genes can be identified in several hours, which is much faster compared to traditional culture method as the real‐time data generation of nanopore sequencer.

Moreover, nanopore sequencing was used to direct sequences in the transcriptome of SARS‐CoV‐2, [ 18 , 19 ] as well as the full‐length coronavirus genomic RNA.[ 20 ]

These studies revealed a complex array of viral transcripts with RNA modifications and provided robust estimates of coronaviral evolutionary rates. Alternatively, considering the relatively low abundance of viral nucleic acids compared to that of host nucleic acids in clinical specimens, direct RNA sequencing and metagenome sequencing methods perform unbiased sequence analysis of both viral and human nucleic acids using a substantial amount of sequencing data and thus resulting in exorbitant associated costs and time to complete the analysis.

Hence, in most recent studies, ARTIC method, based on tiling multiplex PCR and been used to analyze the Zika[ 21 ] and Ebola,[ 22 ] was adopted to get the whole genome of SARS‐CoV‐2 from virus isolates[ 23 ] or clinical samples.[ 24 , 25 , 26 , 27 , 28 , 29 ]

Using this advanced method, by assessing the overlaps among multiplex amplicons, the accurately assembled and complete viral genome can be obtained, which can facilitate the rapid genomic surveillance of SARS‐CoV‐2 for better understanding its pathogenicity, evolution, and transmission.

However, since the large number of clinical samples and the requirement for a short turnaround time, these developed methods are based on nanopore sequencing is not suitable for clinical diagnose and detection of SARS‐CoV‐2.

Importantly, pneumonia and fever can also be caused by other respiratory viruses.[ 30 ] Cross‐infection during the diagnosis process both propagates the spread of SARS‐CoV‐2 and subjects COVID‐19 patients to other respiratory viruses. In severe cases, comprehensive analysis of infecting viruses is necessary.

In addition, although thousands of SARS‐CoV‐2 tests are performed every day around the world, the data obtained by these methods can hardly be used for subsequent analysis of virulence and mutation and for epidemiological investigation. Indeed, nearly all current methods for sequence analysis of the virus are based on whole‐genome sequencing methods, which are costly, with low‐throughput, thereby limiting the data obtained.

However, detection of virulence mutations, virus typing, and epidemiological analysis are critical for the prevention and control of COVID‐19. Therefore, a rapid, accurate, and comprehensive detection method is needed to inform clinical treatment and control cross‐infection to reduce mortality.

Here, we focus on the diagnosis and detection of SARS‐CoV‐2 based on nanopore sequencing since mid‐January 2020, developed a nanopore targeted sequencing (NTS) platform that can combine the advantages of targeted amplification and long‐read, real‐time nanopore sequencing with high sensitivity within 6–10 h and simultaneously detect other respiratory viruses, monitor mutated nucleic acid sequences, and categorize types of SARS‐CoV‐2, and performed NTS for clinical diagnostic tests since early February, 2020.

reference link :https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361204/