Israel Develops Speedy, Precise Coronavirus Test that Circumvents PCR amplification Errors

who fed you in the wilderness with manna, which your fathers had never known, in order to test you by hardships only to benefit you in the end.




(the israel bible)

October 13, 2020

4 min read

For months, there has been a race among scientists around the world to develop the fastest, most accurate and least expensive test to detect SARS-CoV-2, the virus that causes the COVID-19 epidemic. 

The new technique can identify the presence of SARS-CoV-2

Researchers at the Technion-Israel Institute of Technology in Haifa claim to have developed a novel method for rapid and accurate sensing of coronavirus without the need to rely on polymerase chain reaction (PCR) amplification – a method widely used to rapidly make millions to billions of copies of a specific DNA sample. 

 The new technique can identify the presence of SARS-CoV-2 in a sample by counting and quantifying the virus’ RNA molecules with single-molecule precision. Sensing is not biased by PCR amplification errors, permitting future development of a more accurate clinical diagnostic technique.    

The research, which was recently published in the journal ACS Nano under the titleQuantification of mRNA Expression Using Single-Molecule Nanopore Sensing,” was led by Prof. Amit Meller and carried out by postdoctoral researchers Dr. Yana Rozevsky, Dr. Tal Gilboa, Dr. Xander van Kooten, and staff scientist Dr. Diana Huttner – all of whom are researchers in the Faculty of Biomedical Engineering – and Prof. Ulrike Stein and Dr. Dennis Kobelt from the Max Delbrück Center for Molecular Medicine and the Charité Hospital in Berlin.

Developing the most successful test is particularly challenging

RT-qPCR testing, the most widely used test for COVID-19 today, involves a series of preparatory stages, including collecting the sample from a patient using a swab, “opening” the virus and extracting RNA from it. In the next stage, called reverse transcription (RT), specific ‘target’ RNA sequences are copied to the DNA form. Finally, this DNA is amplified by PCR. 

RT-qPCR testing requires large quantities of special reagents, expensive laboratory equipment and highly trained professionals. In addition, recent studies have shown that test results can change from one day to the next and that the massive amplification process can generate significant errors. Developing the most successful test is particularly challenging in cases where the “viral load” (the amount of viral RNA) in a sample is low and can evade sensing. 

This approach opens up the possibility of miniaturizing the diagnostic systems

The new method presented by Meller’s research group relies on original technologies that the lab has developed in the past two decades, using nanofabricated holes (so-called “nanopores”) to sense single biological molecules. The effectiveness of this technology has already been demonstrated in a number of other biomedical uses. Unlike conventional molecular diagnostics, which require large volumes of samples containing millions of copies of the same molecule, nanopore sensing analyzes individual biological molecules from much smaller samples. 

A strong electrical field is used to unfold and thread individual DNA molecules through the nanoscopic hole containing electrical or optical sensors. Each molecule that passes through the hole gives a characteristic “signature,” which enables identification and immediate counting of the molecules. This approach opens up the possibility of miniaturizing the diagnostic systems while improving the accuracy and reliability of tests and expanding the cases where amplifying PCR is not efficient or harms the reliability of the test, the researchers said. 

The new technique successfully quantified the gene’s expression in cancerous cells

The article presents two applications of this method –identifying RNA molecules that signal the emergence of metastatic cancer and detecting coronavirus RNA. To allow unambiguous sensing, the researchers developed a process that leaves only the relevant ‘target’ molecules intact, while degrading all others.

In the first application, the researchers demonstrated the method’s potential for early detection of metastatic cancer by quantifying the levels of MACC1 – one of the primary genes known to signal the formation of a metastatic state. Thanks to its high degree of sensitivity, the new technique successfully quantified the gene’s expression in cancerous cells at the early stages of illness (known as stages I and II) – a challenge that PCR-based technologies failed to meet. Obviously, the earlier these genetic biomarkers are discovered, the better the chances of successful treatment.

In the second application, the researchers detected the RNA molecules of the SARS-CoV-2 virus using the same approach. The technique presented in the article is not the first to analyze single molecules; however, unlike previous reports, it circumvents processes in the sample treatment that introduce “noise” and errors into the system. Two of these processes are sample purification, in which many target molecules are inadvertently lost, and DNA amplification, which may lead to errors and faulty diagnoses. 

The article is an important milestone

“Our system enables quantifiable sensing of the RNA genetic expression levels using a relatively simple nano-sensor device, without needing to cleanse the sample and with no need for massive amplification processes that may harm the test’s sensitivity and reliability,” explained Meller “We have shown that our technology preserves the level of genetic expression of the original RNA molecules throughout the entire process. In this way, we obtain a more precise analysis method, which is essential in both contexts we studied – RNA biomarkers of metastatic cancer and the SARS-CoV-2 virus.”

The article is an important milestone for Meller’s research group, but it is definitely not the end of the road. With further work, the nanopore sensing system is expected to become a portable device that will make cumbersome lab equipment unnecessary. Technological and clinical research is continuing at the Technion Faculty of Biomedical Engineering, in collaboration with the BioBank at the nearby Rambam Medical Center. At the same time, steps are being taken to commercialize the technology in order to make it available for general use as soon as possible. 

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