What Is A Cycle Threshold (Ct) Value In qPCR?
In this article, I will explain what is meant by a Ct value in quantification (real-time) PCR (qPCR). I will also discuss the difference between Ct and Cq values.
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A video tutorial explaining what a Ct value is in qPCR can be found in our Mastering qPCR course.
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The amplification plot
To better understand the Ct value, it is worthwhile looking at the amplification plot of a qPCR run.
Whilst performing a qPCR experiment, the results are commonly displayed on an amplification plot. An example of a typical amplification plot, along with annotations, can be seen below.
The plot resembles a sigmoidal amplification pattern, as indicated by the green line. This green line demonstrates the change in fluorescence over the number of cycles. Typically, in a qPCR experiment, there are 40 cycles. That is 40 rounds of amplification.
Background (or baseline)
At the start of the run, when the amount of PCR product is low, this produces very little fluorescence. This section of cycles (between cycles 0 and 15 in the above figure) is known as the background signal.
Exponential phase
Once the amount of PCR product is amplified enough, it will enter the exponential phase. This is when the amount of PCR product doubles for every PCR cycle. This can be seen in cycles 15 and 25 in the above amplification plot.
Plateau
Once all of the reagents, such as nucleotides, have been used up in the PCR reaction, the amplification will slow and ultimately plateau. This is the region where no more PCR products cannot be produced. This can be seen in cycles 30 and 40 in the above amplification plot.
What is the Ct value?
The cycle threshold (Ct) value of a reaction is defined as the cycle number when the fluorescence of a PCR product can be detected above the background signal. In order to calculate the Ct value, it is necessary to draw a horizontal line (threshold) on the amplification plot. The placement of this line is often determined by the qPCR software, however, users can manually place this line. Ideally, the threshold is placed at the point when the reaction is in the exponential phase, therefore it will not be confused with the background signal.
By looking at the example above, it can be seen that the cycle number when the threshold intersects the amplification plot is 19. Therefore, the Ct value will be 19 in this case.
The Ct value is associated with the amount of PCR product in the reaction. The lower the Ct value, the more PCR product that is present. This is because it takes fewer PCR cycles for that product to be detected over the background signal.
What is the difference between Ct and Cq values?
You will also often hear the term cycle quantification (Cq), maybe even crossing point (Cp) or take-off point (TOP). Basically, these terms all refer to the same value as the Ct value. Discrepancies in nomenclature have been historically inconsistent, with different qPCR manufacturers using different variations. The term Cq was proposed in the Minimum Information for Publication of Quantitative Real-TIme PCR Experiments (MIQE) Guidelines. Therefore, most people actually prefer the term Cq, as opposed to Ct.
Почему метод ПЦР используется в диагностике SARS-CoV-2?
В ответах использованы согласованные позиции Эстонского объединения лабораторной медицины (ELMÜ), Эстонского общества инфекционных болезней (EIS) и Департамента здоровья.
Почему важна ПЦР-диагностика?
Определение РНК вируса SARS-CoV-2 в полученном из дыхательных путей материале с помощью полимеразной цепной реакции в реальном времени (ПЦР) представляет собой т. н. «золотой стандарт» анализа на коронавирус (согласно рекомендациям Всемирной организации здравоохранения [ВОЗ] и других международных организаций, а также в рамках клинической практики).
ПЦР-метод уже годами используется врачами в повседневной практике при диагностике других вирусных инфекций дыхательных путей (например, гриппа).
Можно ли с помощью ПЦР-теста отличить заразных пациентов от незаразных?
ПЦР-тест позволяет выявлять только наличие генетического материала вируса, но не его способность к инфицированию. Определить наличие жизнеспособного вируса можно только при выращивании вируса в культуре клеток, однако для стандартной диагностики этот метод не подходит вследствие его высокой стоимости и длительности проведения (требуется несколько недель).
ПЦР-тест на SARS-CoV-2 не позволяет точно определить количество вируса, однако содержание вируса в полученном у пациента образце можно косвенно оценить с помощью Ct-значения.
Что такое Ct-значение и как оно связано с ПЦР-тестами?
- Ct (cycle threshold) — это количество циклов создания дополнительных копий РНК, начиная с которого результат конкретного теста считается положительным. Ct-значение путают с числом циклов, хотя это два разных значения. Низкое Ct-значение указывает на высокий уровень вируса, которому сопутствует более высокая инфекционность.
- Высокое Ct-значение указывает на низкий уровень вируса в исследуемом материале, что может свидетельствовать о следующем:
- начало инфекционной фазы, когда количество вируса ещё чётко не определяется и человек потенциально уже может заразить окружающих;
- фаза выздоровления (завершения инфекции), если человек до недавнего времени был инфицирован, но на текущий момент риск заражения окружающих уже может быть сниженным.
Ct-значение не указывается как часть результатов теста, оно относится к техническим деталям анализа и не прошло валидацию для использования в рамках клинической практики.
Ct-значения не стандартизированы, поэтому при использовании в лабораториях различных тестов мы можем получить для одного и того же исследуемого материала различные значения. По этой причине сравнение данных значений и какие-либо выводы на их основе не являются клинически обоснованными.
Лаборатории не сообщают Ct-значения во избежание их неправильного толкования. Такой способ действий основывается на консенсусном решении профессиональных обществ (ELMÜ, EIS) и Департамента здоровья.
Как проводится интерпретация результатов анализа на SARS-CoV-2 в лаборатории SYNLAB Eesti?
Используемое в тесте Ct-значение определяется производителем теста на основе результатов исследований.
В таллиннской лаборатории SYNLAB Eesti вирус SARS-CoV-2 определяется по методу ПЦР в реальном времени (RT-PCR) с использованием реактивов ThermoFisher TaqPath COVID-19 CE-IVD RT-PCR. Упомянутые реактивы позволяют выявлять три гена вируса SARS-CoV-2 — N, S и ORF1ab. Согласно установленным производителем правилам, положительным считается результат анализа при подтверждении наличия как минимум двух генов. Помимо Ct-значения, при оценке результата учитываются также показатель RFU (относительная единица флуоресценции) и подъём кривой на графике амплификации.
Более точная информация от производителя ЗДЕСЬ.
Помимо прочего, SYNLAB Eesti успешно осуществила сравнительные испытания с референтной лабораторией ВОЗ, а также регулярно (несколько раз в год) участвует в международных сравнительных испытаниях.
Методика прошла лабораторную верификацию и аккредитацию со стороны Эстонского центра аккредитации. Департамент здоровья выдал лаборатории разрешение на оказание медицинских услуг в рамках специализации на лабораторной медицине.
Как прокомментировать утверждения о том, что большинство положительных результатов ПЦР-теста являются остаточно-положительными, когда пациенты уже не заразны?
Заразность пациента зависит от ряда факторов: количество имеющегося в дыхательных путях вируса, количество выделяемого пациентом вируса (отличается при спокойном дыхании или пении/чихании), соблюдение мер предосторожности (маска, соблюдение дистанции) и т. д. В лаборатории проводят оценку наличия вируса в дыхательных путях. Как объяснено выше, результат ПЦР-теста напрямую не определяет риск передачи инфекции. Опасность заражения окружающих пытались примерно оценить с помощью корреляции Ct-значения и методов выращивания вируса в культуре клеток.
Утверждение, что большая часть положительных результатов ПЦР-теста относится к так называемым остаточно-положительным и пациенты при этом не заразны, может быть верным лишь в определенных ограниченных группах пациентов (например, при проведении скрининга среди случайных людей). Если же говорить о материалах, отправляемых в лабораторию с целью диагностики, то в большинстве случаев (около 90%) у пациентов имеется очень большое количество вируса, что указывает на опасность заражения пациентом окружающих. Лишь менее чем в 5% случаев можно предположить низкую опасность передачи инфекции (на основе данных исследования Jaafar et al. 2020 и статистики по образцам SYNLAB Eesti).
Возможность наличия у пациента положительного результата ПЦР-теста при определённой вероятности отсутствия опасности заражения окружающих не должна использоваться как аргумент для отказа от средств индивидуальной защиты или игнорирования других правил.
Что думают о Ct-значениях врачи-инфекционисты и лабораторные врачи?
Как и в случае с любыми другими анализами, результат теста, в том числе и Ct-значение, следует оценивать с учётом клинического контекста (наличие симптомов, время, прошедшее с момента их появления, а при необходимости также результаты дополнительных анализов, например, на антитела). Решение об опасности заражения пациентом окружающих не может быть автоматическим. Окончательная оценка результата проводится лечащим врачом, который учитывает вышеописанные факторы и принимает решение о необходимости изоляции пациента или о том, что речь может идти об уже перенесённой инфекции.
Важно отметить, что Ct-значения не отличаются у пациентов с симптомами и у пациентов без симптомов. У бессимптомных пациентов сложно точно оценить начало периода, когда они могут заразить окружающих.
MEIE VEEBILEHE PARIMA KASUTAMISE HUVIDES PAIGALDATAKSE TEIE ARVUTISSE “KÜPSISEID”, MIS JÄLGIVAD LEHE KASUTAMIST. VEEBILEHE KÕIKIDE FUNKTSIONAALSUSTE KASUTAMISEKS, KLIKAKE NUPUL “OK”. LINKIDELE VAJUTADES LEIATE LÄHEMAT TEAVET MEIE ANDMEKAITSEPOLIITIKA JA “ KÜPSISTE” POLIITIKA KOHTA.
What do we know about PCR threshold cycles?
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Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.
Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.
Publication
What our experts say
PCR tests are the gold standard for detecting the COVID-19 virus. They are both reliable and accurate. Many types of PCR tests exist. For example, the University of Washington Laboratory offers about six different types. Sometimes, a laboratory can run more than one type of test to confirm a diagnosis.
All PCR tests currently follow the same basic principle. They try to match a piece of the DNA from a test swab (also called a sample) with the same genetic code from part of the SARS-COV-2 virus.
The matching is key to determining if the virus is present. In order to detect the tiny DNA fragments, the PCR machine copies genetic fragments from the sample over and over again to zero in on key elements that confirm or deny a match with the SARS-COV-2 virus. This is a process called a cycle threshold. Each cycle doubles the number of viral RNA molecules from the sample exponentially so each cycle leads to many more of them.
Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.
The “cycle threshold” levels—the amount of zoom ins conducted to detect the virus—vary widely across regions and PCR machines. Several researchers from France, South America, and the United States looked at COVID-19 PCR test results from hospitalized patients. They found that the amount of cycles needed to detect the virus in the patients ranged between 14 and 45.
Cycle thresholds are important for several reasons. Some types of PCR testing, called qualitative PCR tests, indicate whether a sample is positive or negative based on a limited number of cycles. If a manufacturer sets a cycling process to 30, the machine turns off after 30 rounds of copying and searching for SARS-CoV-2. If the machine detects the virus during those 30 rounds, it is reported as a positive test. If none is detected, it is reported as negative.
A different type of PCR test called a quantitative PCR test does not have the same type of limit. These PCR tests help inform public health policies and management like contact tracing, or to help shape clinical treatment plans for patients. Once the quantitative PCR machine detects the viral DNA in the sample, it shuts off, regardless of which cycle it has completed. If it not does detect the viral DNA, it will stop after a certain number of cycles have been run. Quantitative PCR tests might be better at determining whether or not a person who was infected with COVID-19 is still infectious.
Regulatory agencies like the United States Food and Drug Administration gave COVID-19 PCR test manufacturers a lot of flexibility in determining what the qualitative PCR test cycle threshold is to determine whether or not a sample is positive or negative. This is likely because these tests were approved through emergency use authorizations. Traditional, standardized cycle thresholds may not be realistic when fighting off a global pandemic. Many places still require that laboratories performing COVID-19 PCR tests report the cycle threshold numbers of both qualitative and quantitative tests.
There is currently some debate about how many times we should be copying and searching for the virus before determining whether a sample is positive or negative. Some opponents feel that the COVID-19 PCR test is run for too many cycles—in other words, gets copied too many times, thus making it an unreliable indicator of an infection. This group is pushing for the counts to be disclosed for each test so individual physicians, scientists, and lab technicians are able to determine whether or not a test is accurate. Most public health agencies have a requirement for cycle threshold counts for their respective populations, which informs local PCR testing protocols.
Ultimately, there is no standard cycle threshold value that is agreed upon internationally. The U.S. Food and Drug Administration currently gives laboratory manufacturers autonomy in determining how many cycles are needed to determine whether a sample is positive or negative.
Regardless of the number of cycles that can be performed or ct-values, if there is no SARS-COV-2 viral genetic material from the starting sample to amplify, no virus is detected. If one is zooming in on a photo looking for a mosquito, and the mosquito isn’t there, then no amount of zooming in will reveal the bug.
PCR tests are the gold standard for detecting the COVID-19 virus. They are both reliable and accurate. Many types of PCR tests exist. For example, the University of Washington Laboratory offers about six different types. Sometimes, a laboratory can run more than one type of test to confirm a diagnosis.
All PCR tests currently follow the same basic principle. They try to match a piece of the DNA from a test swab (also called a sample) with the same genetic code from part of the SARS-COV-2 virus.
The matching is key to determining if the virus is present. In order to detect the tiny DNA fragments, the PCR machine copies genetic fragments from the sample over and over again to zero in on key elements that confirm or deny a match with the SARS-COV-2 virus. This is a process called a cycle threshold. Each cycle doubles the number of viral RNA molecules from the sample exponentially so each cycle leads to many more of them.
Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.
The “cycle threshold” levels—the amount of zoom ins conducted to detect the virus—vary widely across regions and PCR machines. Several researchers from France, South America, and the United States looked at COVID-19 PCR test results from hospitalized patients. They found that the amount of cycles needed to detect the virus in the patients ranged between 14 and 45.
Cycle thresholds are important for several reasons. Some types of PCR testing, called qualitative PCR tests, indicate whether a sample is positive or negative based on a limited number of cycles. If a manufacturer sets a cycling process to 30, the machine turns off after 30 rounds of copying and searching for SARS-CoV-2. If the machine detects the virus during those 30 rounds, it is reported as a positive test. If none is detected, it is reported as negative.
A different type of PCR test called a quantitative PCR test does not have the same type of limit. These PCR tests help inform public health policies and management like contact tracing, or to help shape clinical treatment plans for patients. Once the quantitative PCR machine detects the viral DNA in the sample, it shuts off, regardless of which cycle it has completed. If it not does detect the viral DNA, it will stop after a certain number of cycles have been run. Quantitative PCR tests might be better at determining whether or not a person who was infected with COVID-19 is still infectious.
Regulatory agencies like the United States Food and Drug Administration gave COVID-19 PCR test manufacturers a lot of flexibility in determining what the qualitative PCR test cycle threshold is to determine whether or not a sample is positive or negative. This is likely because these tests were approved through emergency use authorizations. Traditional, standardized cycle thresholds may not be realistic when fighting off a global pandemic. Many places still require that laboratories performing COVID-19 PCR tests report the cycle threshold numbers of both qualitative and quantitative tests.
There is currently some debate about how many times we should be copying and searching for the virus before determining whether a sample is positive or negative. Some opponents feel that the COVID-19 PCR test is run for too many cycles—in other words, gets copied too many times, thus making it an unreliable indicator of an infection. This group is pushing for the counts to be disclosed for each test so individual physicians, scientists, and lab technicians are able to determine whether or not a test is accurate. Most public health agencies have a requirement for cycle threshold counts for their respective populations, which informs local PCR testing protocols.
Ultimately, there is no standard cycle threshold value that is agreed upon internationally. The U.S. Food and Drug Administration currently gives laboratory manufacturers autonomy in determining how many cycles are needed to determine whether a sample is positive or negative.
Regardless of the number of cycles that can be performed or ct-values, if there is no SARS-COV-2 viral genetic material from the starting sample to amplify, no virus is detected. If one is zooming in on a photo looking for a mosquito, and the mosquito isn’t there, then no amount of zooming in will reveal the bug.
Context and background
There is an ongoing concern on different social media outlets about the usefulness of PCR tests in detecting SARS-COV-2 infections. Some ct-values mentioned by experts have been taken out of context or misunderstood.
Because of this, there has been some debate about how many threshold cycles must be run using PCR tests with different countries, regions, health agencies, and international bodies applying different rates.
There is also a challenge related to PCR tests’ ability to detect very small amounts of the virus. Even inactive viruses that are no longer infectious may show up as positive. The Lancet Microbe recently published a study which suggested that any live virus is no longer in the body nine days after symptoms first began. People are still able to shed the virus for weeks or months after this even if it’s not active or infectious. Thus, having a PCR test that detects viral genetic material doesn’t always mean a person is contagious.
Despite these challenges, PCR tests are reliable even if the ct-values are high and are an excellent tool for diagnosing and detecting the SARS-CoV-2 virus in people.
There is an ongoing concern on different social media outlets about the usefulness of PCR tests in detecting SARS-COV-2 infections. Some ct-values mentioned by experts have been taken out of context or misunderstood.
Because of this, there has been some debate about how many threshold cycles must be run using PCR tests with different countries, regions, health agencies, and international bodies applying different rates.
There is also a challenge related to PCR tests’ ability to detect very small amounts of the virus. Even inactive viruses that are no longer infectious may show up as positive. The Lancet Microbe recently published a study which suggested that any live virus is no longer in the body nine days after symptoms first began. People are still able to shed the virus for weeks or months after this even if it’s not active or infectious. Thus, having a PCR test that detects viral genetic material doesn’t always mean a person is contagious.
Despite these challenges, PCR tests are reliable even if the ct-values are high and are an excellent tool for diagnosing and detecting the SARS-CoV-2 virus in people.
Explained: What is CT value in a Covid-19 test, and how does it correlate with viral load and disease severity?
What is Ct-value in a coronavirus home test? The cycle threshold value emerges in RT-PCR tests for the coronavirus, and determines whether a person is positive for Covid-19 or not. To what extent does it correlate with viral load and disease severity?
CT-Value Covid-19 test: An RT-PCR test detects the virus; Ct value sets a benchmark for whether a person is to be counted as positive. (Express Photo: Amit Mehra)
Among various scientific terms that Covid-19 has made part of the public vocabulary, one is the ‘Ct value’ in RT-PCR tests for determining whether a patient is positive for Covid-19.
This was the subject of a recent request sent by the Maharashtra government to the Indian Council of Medical Research (ICMR). The state sought clarity whether it was advisable to treat a person as Covid-negative if the Ct value is more than 24 and the person is asymptomatic.
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State officials said various ICMR documents had mentioned different Ct values and there were divergent views even among Niti Aayog and the National Centre for Disease Control.
Days later, the ICMR DG wrote back to the state Health Secretary that ICMR has taken inputs from virology laboratories across the country to arrive at a single Ct value cut-off. All patients with a Ct value less than 35 may be considered as positive while those with a Ct value above 35 may be considered as negative, ICMR said.
But what is Ct value?
Short for cycle threshold, Ct is a value that emerges during RT-PCR tests, the gold standard for detection of the SARS-CoV-2 coronavirus. In an RT-PCR test, RNA is extracted from the swab collected from the patient. It is then converted into DNA, which is then amplified. Amplification refers to the process of creating multiple copies of the genetic material — in this case, DNA. This improves the ability of the test to detect the presence of virus. Amplification takes place through a series of cycles — one copy becomes two, two becomes four, and so on — and it is after multiple cycles that a detectable amount of virus is produced.
According to the ICMR advisory, the Ct value of an RT-PCR reaction is the number of cycles at which fluorescence of the PCR product is detectable over and above the background signal. Put simply, the Ct value refers to the number of cycles after which the virus can be detected. If a higher number of cycles is required, it implies that the virus went undetected when the number of cycles was lower. The lower the Ct value, the higher the viral load — because the virus has been spotted after fewer cycles.
Why is CT value important?
To put that in context, let us look at the ICMR advisory and Maharashtra’s letter to ICMR. According to the ICMR, a patient is considered Covid-positive if the Ct value is below 35. In other words, if the virus is detectable after 35 cycles or earlier, then the patient is considered positive. If the benchmark were to be lowered to 24 — the value mentioned in Maharashtra’s letter — it would mean that Ct values in the range 25-34 would not be considered positive. A benchmark of 35, therefore, means that more patients would be considered positive than we would get if the benchmark were 24. The ICMR has said lowering Ct threshold parameter may lead to missing several infectious persons.
One can think of Ct value as a measure of transmission potential, said leading virologist Dr Shahid Jameel. “So if there is more virus in my throat and nose, I will transmit it better,” said Dr Jameel, who is Director of the Trivedi School of Biosciences at Ashoka University.
What is the significance of the ICMR threshold of 35?
Globally, the accepted cut-off for Ct value for Covid-19 ranges between 35 and 40, depending on instructions from the respective manufacturers of testing equipment. The ICMR has arrived at the Ct value of 35 based on laboratory experiences and inputs taken from several virology labs.
There was no new advisory, but ICMR informed the Maharashtra government that it was not advisable to use a lower cycle threshold parameter as it would lead to missing several infectious persons and increase disease transmission, Dr Balram Bhargava, DG of ICMR, has said.
Is there any correlation between a Ct value and severity of disease?
No. Although Ct value is inversely correlated with viral load, It does not have any bearing on the severity of the disease, experts have said. A patient can have a low Ct value, which means her viral load is high enough to be detected rapidly, but she may still be asymptomatic.
A small study published in the Indian Journal of Medical Microbiology in January this year found that there was no correlation between Ct values and severity of disease or mortality in patients with Covid-19 disease. It found that the time since the onset of symptoms has a stronger relationship with Ct values as compared to the severity of the disease.
The Ct value tells us about the viral load in the throat and not in the lungs, said Dr Parikshit Prayag, consultant for infectious diseases at Deenanath Mangeshkar Hospital, Pune. “The Ct value does not correlate with severity – only with infectivity. In the first report I do not really look at the Ct value, but for follow -up of patients in the hospital, I do consider the Ct value, as then I can decide whether to transfer the patient to the non-Covid building or not. From the infectivity point of view, it can matter, not severity,” Dr Prayag said.
Does a high Ct value always mean a low viral load?
While that may be the obvious inference, some experts stress that some patients can have a high Ct value and yet have a very significant level of Covid-19 infection, and vice versa. Many factors are important in interpreting an RT-PCR test, and the results may also depend on the method of specimen collection and time from infection to collection and to analysis.
An ICMR advisory in August last year noted that Ct values depend on how the sample has been collected. A poorly collected sample may reflect inappropriate Ct values. Besides, Ct values are also determined by the technical competence of the person performing the test, calibration of the equipment, and the analytical skills of the interpreters.
Again, Ct values may differ between nasal and oropharyngeal specimens collected from the same individual. The temperature of transportation, as well as the time taken from collection to receipt in the lab, can also adversely impact Ct values.