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SARS-CoV-2 testing: methods and problems
The document outlines the procedures and challenges of testing for SARS-CoV-2, emphasizing the necessity of converting RNA to DNA through reverse transcription and utilizing PCR for amplification. It discusses the various modifications of tests, shortages due to bureaucratic red tape, and issues with the original CDC test kit that hampered effective testing. Improvements in testing protocols are underway as more entities are permitted to develop and deploy their own test kits.
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SARS-CoV-2 testing: methods and problems
- 1. Testing for SARS-CoV-2: Methodsand Problems Rachel Mackelprang, Ph.D. California State University Northridge Department of Biology rachel.mackelprang@csun.edu @rmackelprang
- 2. How do wetest for SARS-CoV-2? SARS-CoV-2 has a RNA genome (rather than DNA, which all non- viral organisms have). To perform the test, first RNA must be converted to DNA. This process is called reverse transcription
- 3. How do wetest for SARS-CoV-2? Nasopharyngeal swab SARS-CoV-2 has a RNA genome (rather than DNA, which all non- viral organisms have). To perform the test, first RNA must be converted to DNA. This process is called reverse transcription
- 4. How do wetest for SARS-CoV-2? Nasopharyngeal swab SARS-CoV-2 has a RNA genome (rather than DNA, which all non- viral organisms have). To perform the test, first RNA must be converted to DNA. This process is called reverse transcription Sample collection tube Place sample into collection tube
- 5. How do wetest for SARS-CoV-2? Nasopharyngeal swab SARS-CoV-2 has a RNA genome (rather than DNA, which all non- viral organisms have). To perform the test, first RNA must be converted to DNA. This process is called reverse transcription Sample collection tube RNA Place sample into collection tube Extract RNA
- 6. How do wetest for SARS-CoV-2? Nasopharyngeal swab SARS-CoV-2 has a RNA genome (rather than DNA, which all non- viral organisms have). To perform the test, first RNA must be converted to DNA. This process is called reverse transcription Sample collection tube RNA DNA Place sample into collection tube Extract RNA Reverse transcribe RNA to DNA
- 7. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed.
- 8. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed. PCR amplification is a multi-step process. Each round of amplification doubles the number of DNA molecules
- 9. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed. PCR amplification is a multi-step process. Each round of amplification doubles the number of DNA molecules 1st round
- 10. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed. PCR amplification is a multi-step process. Each round of amplification doubles the number of DNA molecules 1st round 2nd round
- 11. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed. PCR amplification is a multi-step process. Each round of amplification doubles the number of DNA molecules 1st round 2nd round 3rd round
- 12. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed. PCR amplification is a multi-step process. Each round of amplification doubles the number of DNA molecules 1st round 2nd round 3rd round 4th round
- 13. How do wetest for SARS-CoV-2? The next step uses a DNA amplification technique called PCR. In PCR, minute amounts of DNA is turned into large enough quantities to be analyzed. PCR amplification is a multi-step process. Each round of amplification doubles the number of DNA molecules 1st round 2nd round 3rd round 4th round After 40 rounds of amplification, there are billions of copies
- 14. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA
- 15. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA
- 16. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA First round
- 17. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA First round Second round
- 18. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA First round Second round Third round
- 19. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA First round Second round Third round 40th round Billions of copies
- 20. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA First round Second round Third round 40th round Billions of copies In the beginning, there is little to no fluorescent signal
- 21. Testing uses amodification of PCR, called RT-PCR In RT-PCR, DNA is combined with a fluorescent dye that glows in the presence of SARS-CoV-2 DNA Starting DNA First round Second round Third round 40th round Billions of copies In the beginning, there is little to no fluorescent signal Increasing fluorescent signal indicates a positive test: the individual is infected with SARS-CoV-2
- 22. The news reportsthat there are multiple versions of the test—what does that mean?
- 23. The news reportsthat there are multiple versions of the test—what does that mean? Conceptually, different test versions are similar. They use RT-PCR to detect the presence of the SARS-CoV-2 genome.
- 24. The news reportsthat there are multiple versions of the test—what does that mean? Conceptually, different test versions are similar. They use RT-PCR to detect the presence of the SARS-CoV-2 genome. One way they differ is in which part of the SARS-CoV-2 genome they detect.
- 25. The news reportsthat there are multiple versions of the test—what does that mean? Conceptually, different test versions are similar. They use RT-PCR to detect the presence of the SARS-CoV-2 genome. One way they differ is in which part of the SARS-CoV-2 genome they detect. This is a representation of the SARS-CoV-2 genome. It is ~32,000 bases (A,C,U,G nucleotides) long. Each gene is shown by a differently colored box.
- 26. The news reportsthat there are multiple versions of the test—what does that mean? Conceptually, different test versions are similar. They use RT-PCR to detect the presence of the SARS-CoV-2 genome. One way they differ is in which part of the SARS-CoV-2 genome they detect. This is a representation of the SARS-CoV-2 genome. It is ~32,000 bases (A,C,U,G nucleotides) long. Each gene is shown by a differently colored box. Each black bar is a piece of the genome that can be used for testing. Which genome pieces are actually used varies by country.
- 27. The news reportsthat there are multiple versions of the test—what does that mean? Conceptually, different test versions are similar. They use RT-PCR to detect the presence of the SARS-CoV-2 genome. One way they differ is in which part of the SARS-CoV-2 genome they detect. This is a representation of the SARS-CoV-2 genome. It is ~32,000 bases (A,C,U,G nucleotides) long. Each gene is shown by a differently colored box. Each black bar is a piece of the genome that can be used for testing. Which genome pieces are actually used varies by country. Tests also vary by which chemicals are used to perform the tests.
- 28. Why is therea shortage of tests? There is no single reason. Instead, there are multiple factors https://www.nytimes.com/2020/03/10/us/coronavirus-testing-delays.html
- 29. Why is therea shortage of tests? There is no single reason. Instead, there are multiple factors The CDC developed its own version of the test (instead of using WHO protocols), which had problems. https://www.nytimes.com/2020/03/10/us/coronavirus-testing-delays.html
- 30. Why is therea shortage of tests? There is no single reason. Instead, there are multiple factors The CDC developed its own version of the test (instead of using WHO protocols), which had problems. Red tape prevented university, local and state government, and commercial labs from developing and using their own test kits. https://www.nytimes.com/2020/03/10/us/coronavirus-testing-delays.html
- 31. Why is therea shortage of tests? There is no single reason. Instead, there are multiple factors The CDC developed its own version of the test (instead of using WHO protocols), which had problems. Most states had to send their samples to the CDC until March 2nd instead of running the tests in local labs. Red tape prevented university, local and state government, and commercial labs from developing and using their own test kits. https://www.nytimes.com/2020/03/10/us/coronavirus-testing-delays.html
- 32. Why is therea shortage of tests? There is no single reason. Instead, there are multiple factors The CDC developed its own version of the test (instead of using WHO protocols), which had problems. Most states had to send their samples to the CDC until March 2nd instead of running the tests in local labs. There is a shortage of chemicals used to conduct the test Red tape prevented university, local and state government, and commercial labs from developing and using their own test kits. https://www.nytimes.com/2020/03/10/us/coronavirus-testing-delays.html
- 33. Why is therea shortage of tests? There is no single reason. Instead, there are multiple factors The CDC developed its own version of the test (instead of using WHO protocols), which had problems. Most states had to send their samples to the CDC until March 2nd instead of running the tests in local labs. There is a shortage of chemicals used to conduct the test Red tape prevented university, local and state government, and commercial labs from developing and using their own test kits. Details explaining these factors are in the following slides https://www.nytimes.com/2020/03/10/us/coronavirus-testing-delays.html
- 34. What were theproblems with the original CDC kit? The kit was supposed to run three tests. Two of the three were specific to SARS-CoV-2. The third would match many types coronaviruses*. Expected results: Infected individual Test 1 Test 2 Test 3 Infected with another coronavirus Uninfected *There are 4 coronaviruses that generally infect humans, causing ~20% of all common colds. SARS and MERS are also coronaviruses
- 35. What were theproblems with the original CDC kit? Labs that received the CDC kit began testing them and many found that the kits did not work as intended Expected results: Test 1 Test 2 Test 3 Uninfected Actual results: Test 1 Test 2 Test 3 Uninfected
- 36. What were theproblems with the original CDC kit? Labs that received the CDC kit began testing them and many found that the kits did not work as intended Expected results: Test 1 Test 2 Test 3 Uninfected Actual results: Test 1 Test 2 Test 3 Uninfected
- 37. What were theproblems with the original CDC kit? Labs that received the CDC kit began testing them and many found that the kits did not work as intended Expected results: Test 1 Test 2 Test 3 Uninfected Actual results: Test 1 Test 2 Test 3 Uninfected When one of the components doesn't work, it brings the validity of the entire test into question.
- 38. What were theproblems with the original CDC kit? Labs that received the CDC kit began testing them and many found that the kits did not work as intended Expected results: Test 1 Test 2 Test 3 Uninfected Actual results: Test 1 Test 2 Test 3 Uninfected This caused a multi-week delay in getting working kits into the hands of testing labs. When one of the components doesn't work, it brings the validity of the entire test into question.
- 39. What were theproblems with the original CDC kit? Labs that received the CDC kit began testing them and many found that the kits did not work as intended Expected results: Test 1 Test 2 Test 3 Uninfected Actual results: Test 1 Test 2 Test 3 Uninfected This caused a multi-week delay in getting working kits into the hands of testing labs. Lack of tests make it impossible to get an accurate picture of how far and fast the disease is spreading When one of the components doesn't work, it brings the validity of the entire test into question.
- 40. Why didn’t theCDC use the WHO test protocol? It’s hard to say why the administration declined to used the WHO protocol, even as a temporary bridge until it could develop its own. Rachel here—creator of this slide deck: I couldn’t find any good information beyond speculation to answer this question. If anyone reading this has an answer (and can provide a reliable source), please contact me. rachel.mackelprang@csun.edu
- 41. Red Tape Labs allover the world use PCR to answer myriads of research questions. But only certain tests and techniques can be used to diagnose disease. https://jamanetwork.com/journals/jama/fullarticle/2762951 https://www.newyorker.com/news/news-desk/what-went-wrong-with-coronavirus-testing-in-the-us
- 42. Red Tape Labs allover the world use PCR to answer myriads of research questions. But only certain tests and techniques can be used to diagnose disease. Normally, this is a good way to ensure proper testing. It reduces the number of misdiagnoses. https://jamanetwork.com/journals/jama/fullarticle/2762951 https://www.newyorker.com/news/news-desk/what-went-wrong-with-coronavirus-testing-in-the-us
- 43. Red Tape Labs allover the world use PCR to answer myriads of research questions. But only certain tests and techniques can be used to diagnose disease. Normally, this is a good way to ensure proper testing. It reduces the number of misdiagnoses. In a pandemic this can lead to a shortage of tests. In an emergency, the FDA can authorize the use of unapproved medical products and tests. Labs wanting to runs tests can submit an Emergency Use Authorization (EUA) request. https://jamanetwork.com/journals/jama/fullarticle/2762951 https://www.newyorker.com/news/news-desk/what-went-wrong-with-coronavirus-testing-in-the-us
- 44. Red Tape Labs allover the world use PCR to answer myriads of research questions. But only certain tests and techniques can be used to diagnose disease. Normally, this is a good way to ensure proper testing. It reduces the number of misdiagnoses. In a pandemic this can lead to a shortage of tests. In an emergency, the FDA can authorize the use of unapproved medical products and tests. Labs wanting to runs tests can submit an Emergency Use Authorization (EUA) request. Actually getting an EUA proved to be difficult, frustrating, and time consuming, leading to further delays. https://jamanetwork.com/journals/jama/fullarticle/2762951 https://www.newyorker.com/news/news-desk/what-went-wrong-with-coronavirus-testing-in-the-us
- 45. Red Tape Labs allover the world use PCR to answer myriads of research questions. But only certain tests and techniques can be used to diagnose disease. Normally, this is a good way to ensure proper testing. It reduces the number of misdiagnoses. In a pandemic this can lead to a shortage of tests. In an emergency, the FDA can authorize the use of unapproved medical products and tests. Labs wanting to runs tests can submit an Emergency Use Authorization (EUA) request. Actually getting an EUA proved to be difficult, frustrating, and time consuming, leading to further delays. https://jamanetwork.com/journals/jama/fullarticle/2762951 https://www.newyorker.com/news/news-desk/what-went-wrong-with-coronavirus-testing-in-the-us Improvements are being made. For example, the FDA will now allow private companies to produce test kits, which will be approved retroactively.
- 46. When will wehave enough tests? This is a hard question to answer. Much of the red tape has been reduced. Universities, private companies, and state/local governments are now developing and deploying their own tests.
- 47. You cannot fighta fire blindfolded "We have a simple message for all countries: test, test, test.” “The most effective way to prevent infections and save lives is breaking the chains of transmission. And to do that, you must test and isolate. You cannot fight a fire blindfolded. And we cannot stop this pandemic if we don't know who is infected.” Message from WHO Director General Dr. Tedros