PCR Thermal Cyclers: How is the COVID-19 Virus Detected?

The number of confirmed global deaths from COVID-19 is almost 4.2 million as of July 2021, according to a Statista report. By comparison, the last Great Plague of 1665 killed a million people. The number of fatalities due to the novel coronavirus is a staggering figure, especially considering that we are in an age of revolutionary medical discoveries and technology.

Since the 2019 discovery of SARS-CoV-2 or COVID-19 in China, scientists have successfully identified the novel virus. At present, several countries have also developed COVID-19 vaccines, and mass vaccination campaigns are underway.

However, detecting the COVID-19 virus was critical to making this happen. It was the pathway that led to the eventual development of the vaccine. In the beginning, scientists used a combination of whole-genome sequencing, computed tomography imaging, electron microscopy, and other diagnostic tools to identify SARS-CoV-2.

PCR, short for polymerase chain reaction, is one of the most widely used methods for identifying the COVID-19 virus. This post breaks down the science behind this method of identifying the novel coronavirus and identifies the best PCR thermal cyclers on the market.

What are PCR Thermal Cyclers?

The primary fields where PCR thermal cyclers are indispensable are:

• Immunology
• Oncology
• Human Genome Project
• Population biology
• Researches in zoology and botany
• Forensics
• Paleontology
• Hereditary disease and tumor
• Clinical diagnosis of viruses

PCR thermal cyclers allow scientists and researchers to make millions of copies of a specific section of DNA and RNA from the smallest sample. For DNA and RNA sequencing, PCR makes it easy to detect the absence or presence of genes to identify pathogens.

The first commercial thermal cycler, TC1 DNA, hit the market in 1987. The first thermal cycler was rather basic, although it could heat and cool samples with a metal block. However, modern PCR thermal cyclers have come a long way with significant advancements in technology and functions.

Some of the best PCR thermal cyclers on the market come with precise temperature control and multiple block formats. In addition, you can also run two independent experiments simultaneously, which increases the output in a significant manner. 

At present, there are many types of thermal cycles pcr, each with different features – from personal to mini thermal cyclers that easily fit on a desk to larger ones. In addition, you can also interlink several thermal cyclers to form a larger PCR system, especially in extensive projects. A large PCR system can also be easily hooked up to a central computer for automation and control.

How is the COVID-19 virus detected using PCR thermal cyclers?

Among the three tests employed to detect SARS-CoV-2, PCR is considered the most reliable in terms of results. COVID-19 PCR test results are also the most accurate for detecting active coronavirus infection.

The section below details how PCR thermal cyclers detect SARS-CoV-2.

Sample collection

The steps for detecting the SARS-CoV-2 virus begin with collecting samples from a host, usually a human. Typically, sample collectors employ two types of swabs for COVID testing.

First is the nasal swab, which collects a sample from the nostrils and the periphery. The second is the nasopharyngeal swab, in which a flexible sample-collection tip is inserted into the nasal cavity.

In some cases, COVID swabs are also collected from the throat. But it is less common than nasal swabs. For scientific research purposes, the collection of stool, serum, and ocular secretions is not uncommon.

In addition, some laboratories also rely on self-collected saliva samples. Saliva samples are relatively painless compared to upper respiratory tract sample collection. This method also enables higher-volume testing in labs and reduces the risk of contamination for healthcare providers.

The technician or the medical person who collects the sample seals it in a test tube and sends it to the laboratory for testing.

Extraction

The technician isolates genetic material in the lab – the RNA of the potential virus from the sample. The technician typically treats the sample with chemical solutions to remove substances, including fats and proteins.

After the extraction is complete, the RNA from the sample is isolated. This RNA is a mixture of the host’s genetic material and the SARS-CoV-2 virus.

PCR or Polymerase Chain Reaction

Polymerase Chain Reaction is the stage that detects the presence of the SARS-CoV-2 virus in the sample. The isolated RNA is mixed with a special ingredient to start the PCR process, typically enzymes. Special enzymes usually vary according to the different viruses that are being tested. 

Therefore, for SARS-CoV-2, the key enzymes are reverse transcriptase and DNA polymerase. In addition, other ingredients include:

• DNA building blocks.

• Probes or marker labels.

• Cofactors.

• Primers that identify and bind easily to the SARS-CoV-2.

Like using a special enzyme to detect the COVID-19 virus, specific probes and primers are also required. According to the CDC, some of the recommended probes and primers for the detection of COVID-19 include:

• Forward primers – 2019-nCoV_N1-F, 2019-nCoV_N2-F, RP-F

• Reverse primers – 2019-nCoV_N1-R, 2019-nCoV_N2-R, RP-R

• Probes – 2019-nCoV_N1-P, 2019-nCoV_N1-P, 2019-nCoV_N2-P, 2019-nCoV_N2-P, RP-P, RP-P

This list of the COVID-19 testing probes and primers is by no means exhaustive or absolute. SARS-CoV-2 is a novel coronavirus – meaning that no known tests about the virus existed before 2020. Besides, the global infection rates were also exploding without control. This prompted scientists and researchers to develop the primers, probes, and even testing methods for SARS-CoV-2 from scratch and in minimal time.

Therefore, in addition to using the other primers and probes mentioned above, the testing methods may also be tweaked to obtain the most accurate results for detecting the COVID-19 virus. Furthermore, primers and probes for testing coronavirus samples also vary from one country to the other.

Visit this link for a full list of primers and probes used in other countries, including Germany, Japan, and China. 

Note: Combining the RNA with these ingredients is essential as it converts the RNA into DNA. Conversion of isolated SARS-CoV-2 RNA into DNA copy is a vital process since PCR only works for DNA. 

Along with the enzymes, the SARS-CoV-2 DNA is denatured into short primers at high temperatures. Simultaneously, these short complementary DNA primer templates come together and stick, forming the base site for DNA synthesis. As the DNA continues to join, the short primers extend to form a copy of the SARS-CoV-2 DNA.

Furthermore, the second primer also forms on the opposite orientation of the first primer. This results in the formation of a SARS-CoV-2 DNA copy that is complementary to the original strand. The next round of heating produces further copies of the SARS-CoV-2 DNA strands, and the cycle is repeated until enough copies of COVID-19 DNA copies are amplified.

Regardless of whether the extracted RNA is from a COVID-19 sample or not, the polymerase chain reaction follows the same principle and involves three stages. These are denaturing, annealing, and extending.

Many important processes occur during the three stages of PCR, as detailed in the following section.

Denaturing

It is the first stage of the process of polymerase chain reaction. Here, isolated SARS-CoV-2 RNA is mixed with specific enzymes and other components.

This cocktail is put inside a PRC thermal cycler that heats up to 95oC or 203oF. The combination of extremely high temperature and specific enzymes separates the hydrogen bonds between the two strands of the SARS-CoV-2 DNA template.

This separation of the SARS-CoV-2 DNA into two strands forms the DNA templates where the production of new COVID-19 strands will occur. The whole process of DNA strand separation completes in about 15 to 30 seconds. However, maintaining a constant 94 to 95oC temperature is critical for the successful completion of denaturing.

In this regard, using a PCR thermal cycler that is great in quality is vital. Substandard thermal cyclers PCR will not have the features or the capacity to withstand extremely high temperatures.

Annealing

The second stage is annealing, where the high temperature is cooled down to about 50 to 65oC or 122 to 149oF. As cooling occurs, the primers and probes bind to complementary regions, forming template DNA strands via hydrogen bonding. This binding process results in the formation of two-stranded DNA. It also allows the DNA polymerase to form the complementary strands from the loose base templates or the building blocks.

In detecting the SARS-CoV-2 virus, DNA polymerase, a specialized enzyme, begins assembling new strands from the template DNA. The newly formed DNA strands run in opposite directions, thereby forming the forward and the reverse primer. In addition, the new strands are complementary, allowing amplification of the viral DNA.

A standard PCR thermal cycler doubles the number of DNA copies. So one cycle makes two DNA copies, and the second cycle makes four copies, etc. Most PCR thermal cyclers can run up to 35 cycles, making up to 35 billion DNA copies by the end of all the cycles.

The annealing stage in SARS-CoV-2 detection takes about 10 to 30 seconds.

Extending

Extending is the final stage in the polymerase chain reaction. In the extending stage, the temperature of the PCR thermal cycler is taken up to 72oC or 161oF. It involves the formation of new DNA strands by the addition of bases by an enzyme.

In most cases of virus detection, Taq DNA polymerase is used. This DNA polymerase comes from a bacterium that lives in hot springs and can withstand extremely high temperatures.

The Taq DNA polymerase starts building the new DNA strand by attaching it to the primer. In addition, the polymerase enzyme adds nucleotides to the single DNA strands in both the forward and reverse directions relative to the primer.

At the end of the extending cycle is a new DNA strand complete with the double molecule structure. The duration of the extension stage typically depends on the length of the viral DNA being sequenced. However, a good PCR thermal cycler can generally copy up to 1,000 DNA copies in under a minute.

The lab technician can also tell whether SARS-CoV-2 is present in the sample around the time the DNA copies become available in the PCR thermal cycler. Simultaneously as the cycler makes new copies of DNA, the primers also start copying the targeted sections in the complementary strands.

Furthermore, the probes or marker labels start attaching themselves to the newly formed DNA fragments and visually signals the thermal cycler’s screen. The signal is usually a fluorescent dye that the cycler can detect easily. The PCR thermal cycler’s internal computer measures the frequency and amount of these visual signals in real time. Therefore, thermal cyclers are also known as RT-PCR.

The cycler’s internal computer monitors the fluorescent dye signal in each cycle to confirm the presence of SARS-CoV-2 in the sample. If fluorescent probes exceed a certain threshold, it indicates that the nasal sample contains SARS-CoV-2 (the COVID-19 virus).

In addition, the scientist or lab technician monitors the number of cycles required for the probes to reach the limit set during testing. For each COVID-19 sample, the fewer PCR cycles it takes to reach the level to determine the severity of the coronavirus infection in the COVID-19 positive patient.

On the other hand, if SARS-CoV-2 is not present in the sample, the probes will not bind to the newly synthesized DNA fragments. In addition, there will be no visual signal on the thermal cycler’s screen. In this case, the COVID-19 sample is declared SARS-CoV-2 virus-free, and the test is negative.

What is the use of a thermal cycler in PCR?

Thermal cyclers, PCR machines, or thermocyclers are indispensable devices in the polymerase chain reaction. The main function of a thermal cycler is to amplify DNA and RNA samples through the polymerase chain reaction. Thermal cyclers also allow the seamless transition between extremely high and low temperatures, vital in the three stages of PCR.

That being, the main uses and features of the thermal cycler in PCR are as follows:

Heating and cooling

Thermal cyclers used in PCR seamlessly heat and cool DNA or RNA samples. Depending on the brand and model of the thermal cycler, it can reach a maximum temperature of 95 °C (203 °F).

In addition, the lid temperature of thermal cyclers can withstand extreme heat up to 105 °C (221 °F). The cycler’s lid’s ability to withstand such high temperatures prevents the DNA or RNA sample in the centrifuge tube from evaporating.

The heated lid of a thermal cycler also prevents water condensation on the inside of the lid, which could compromise the final test results. In older thermal cyclers, mineral oil or paraffin was used for this purpose.

Temperature ramp control

A thermal cycler is also useful for controlling temperature ramps. Because the machine undergoes extreme heat and cooling differences, the automatic ramp settings easily control the temperature without difficulties.

Some of the best thermal cyclers, PCR, have a ramp time feature. Ramp time is the duration it takes for the machine to heat up or cool down to a specific temperature. In addition, you can program the thermal cycler to run at a specific temperature for the desired number of cycles and time.

Auto-restart function

In the event of a power failure during a chain reaction, thermal cyclers can easily auto-restart. The auto-restart feature in a thermal cycle is significant as it allows the machine to restore the data and continue the cycle without interruption. Without this function, PCR testing for COVID-19 samples will not be as quick.

Reliable operation

Thermal cyclers are also equipped with a thermoelectric module for thermoelectric cooling. It is due to these features in thermal cyclers that PCR can operate reliably under stable conditions.

The earliest PCR thermal cyclers were less reliable, compromising test results. But modern thermocyclers on the market are extremely reliable and have become the gold standard for SARS-CoV-2 testing.

Real-time display and function

Thermal cyclers, PCR can also monitor the polymerase chain reaction in real-time, allowing researchers to read the test results instantly. This enables COVID-19 test results to be available within 24 hours of nasal sample collection.

In addition, the cycler inspects the execution and remaining execution time of each cycle in the reaction.

Multiple functions

Advantages of using PCR thermal cyclers for the detection of the COVID-19 virus

1. Accurate results

Since the discovery of SARS-CoV-2 in Dec 2019, PCR Thermocyclers have proven to be the gold standard for detecting the virus. According to a January 2021 study, the accuracy of PCR tests in detecting COVID-19 is as high as 99.7%. 

The accuracy of the PCR tests varies slightly depending on whether the sample is sputum, urine, stool, rectal swab, or plasma. Nevertheless, PCR thermal cycler testing for SARS-CoV-2 remains the most accurate method to date. 

2. Quick procedure and fast results

Another outstanding strength of using the polymerase chain reaction to test for SARS-CoV-2 is how quickly it is. Depending on the number of COVID-19 cases being tested, all the cycles in the reaction can be completed in as little as two hours. This allows the labs to release the test results almost instantly.

In addition, thermal cyclers can accelerate DNA amplification. Within a couple of hours, the machine can make millions and billions of copies. If the testing lab or the agency incorporates robotic assistance and automation, the process can be even quicker. 

Setting up the PCR Thermocyclers is also quick. The thermal cycler can be set up and running in a matter of minutes, especially if the machine is high-quality. 

3. Requires very small samples

The PCR thermocycler method also requires very few samples from COVID-19 patients. This is a great advantage, as health care workers can collect a large number of samples in a short time. A swab from the respiratory tract of a potential coronavirus patient provides enough DNA/RNA to generate millions of copies. 

A drawback of using PCR thermal cyclers for the detection of the COVID-19 virus

PCR thermal cyclers have been revolutionary in helping laboratories and testing agencies to detect the COVID-19 virus. Despite their outstanding and invaluable utility, there are a couple of drawbacks to using PCR thermal cyclers for SARS-CoV-2 detection. 

Low supply

Testing agencies in many countries rely on the PCR thermal cyclers to get the most accurate testing of COVID-19 samples. The availability and supply of PCR thermal cyclers are insufficient relative to rising demand. And because the quality of thermal cyclers is paramount to testing results, high-quality PCR thermal cyclers are in short supply. 

The shortage of PCR thermal cyclers is one of the main reasons many provinces in China resorted to CT scans for clinical diagnosis of COVID-19. However, the CT scan results for SARS-CoV-2 were unreliable. It also had a very high rate of negative COVID-19 results, which is one of the main drawbacks. 

Another grave drawback of using CT scans to detect the SARS-CoV-2 virus is their low specificity. It is because the imaging results from CT scans often overlap with other respiratory infections, particularly pneumonia. 

Lack of PCR thermal cycler infrastructure

Not all laboratories and hospitals across the globe are in urban locations with state-of-the-art infrastructure. Community hospitals and other public health centers outside urban areas lack the necessary infrastructure to perform PCR. 

In addition, even community hospitals that have PCR thermal cyclers lack trained personnel or technicians to operate them. 

PCR thermal cyclers rely on detectable SARS-CoV-2 virus

Because thermal cyclers rely on detectable SARS-CoV-2 virus, they can produce rapid results while testing the sample. However, the caveat is that the thermal cycler does not detect the SARS-CoV-2 virus in asymptomatic patients. 

This is especially dangerous if the asymptomatic COVID-19 patient has recovered. Because PCR testing does not detect SARS-CoV-2 in such a patient, appropriate preventive measures will have been avoided, and the person can become a super-spreader. 

What are the Best PCR Thermocyclers?

In this section, we review the top PCR Thermocyclers for running the polymerase chain reaction. 

1. Applied Biosystems 7500 ($9,999.00)

The 7500 series PCR thermal cycler from Applied Biosystems is among the top devices for detecting SARS-CoV-2. The 5-color fluorescence signal system is undeniably among its best features. In addition, the 7500 thermal cyclers come with a 96-well format that makes setting up effortless, even without automation.  

Another great feature in the 7500 thermal cyclers is the provision to accommodate tube strips. This allows the technician to cap the pipettes immediately after filling them with samples, speeding up the entire process. High-resolution melting analysis is also an indispensable feature of PCR. 

The Applied Biosystems 7500 series is available in three models: 7500 Fast RT PCR System, 7500 Fast Dx RT PCR System, and 7500 RT PCR System. There are a few differences in features between the three models, but each yields speedy results. In addition, the three PCR models in the 7500 series come with either a laptop or a tower computer kit. 

2. SimpliAmp Thermal Cycler ($2,250.00)

SimpliAmp is another thermal cycler from Applied Biosystems. This thermal cycler is one of the most compact on the market. But despite its small size, it runs with precision and enables accurate optimization. 

One of the outstanding features of the SimpliAmp thermal cycler is its large, intuitive touchscreen. It enables simulation modes and makes programming very easy.

Simulation modes in a thermal cycler are a beneficial feature if you want to transition from an existing or older thermal cycler to the new one, such as the SimpliAmp.

SimpliAmp does not come with a computer. However, the thermal cycler can be connected to a desktop or laptop via software.

3. ProFlex PCR System ($5,640.00)

ProFlex PCR System is another thermal cycler from Applied Biosystems. It has a compact design but is packed with top features.

The five interchangeable block formats are among its best features, as you can run three experiments concurrently. 

Another great feature of the ProFlex PCR System is the ease of interchanging the blocks at the flick of a switch. The dual-block setting with 96-well and 384-well plates is another feature that yields high sequencing output.The 

ProFlex PCR System does not come with a computer but is compatible with computers and smartphones. 

4. Roche LightCycler 480 II ($7,899.00)

The LightCycler 480 is a high-performance PCR thermal cycler. It is among the most versatile thermal cyclers, allowing you to perform medium- and high-throughput reactions. 

The thermal cycler also supports mono- and multicolored tests, which is an outstanding feature. Furthermore, the 96-well and 384-well block settings allow multiple-plate analysis with ease. In addition, the LightCycler 480 comes with a wide set of probes and provisions for fluorescent dyes. 

5. Abbott ID NOW ($2,999.00)

ID NOW from Abbott is viral for its quick results. Depending on the sample tested, ID NOW can produce results in less than 13 minutes, which is a great achievement. ID NOW was formerly called Alere-i.

This Abbott device is also very portable and indispensable for rapid COVID-19 testing, especially in the US. Some of the top features of ID NOW are bi-directional connectivity, QC Lockout, and near-patient testing. Near-patient testing not only reduces testing and healthcare costs but also makes this device one of the best molecular point-of-care platforms for COVID-19 testing.

6. Cobas Liat ($8,900.00)

With a compact and portable design, Cobas Liat is a premium solution for many healthcare facilities. In addition to its compact design, Cobas Liat delivers rapid results, making it a perfect PCR point-of-care solution for COVID-19 detection. 

Apart from its compact design and portability, it takes about 20 minutes or less to obtain test results with the Cobas Liat. In addition, the results of the SARS-CoV-2 test are highly inaccurate, which affects testing at clinics and satellite locations. 

Other features of the Cobas Liat worth mentioning include fully automated operation and an intuitive user interface. In addition to SARS-CoV-2, this PCR solution is ideal for testing other respiratory infections, such as influenza, with high accuracy. 

7. Bio Rad C1000 ($2,490.00)

The large touchscreen on the Bio-Rad C1000 is what stands out at first glance. The touchscreen makes navigation and protocol editing straightforward. This modular PCR platform offers superior performance for SARS-CoV-2 virus testing. 

The interchangeable reactions module, which swaps effortlessly without any tools, is one of its best features. Another great feature of the Bio-Rad C1000 is its protocol programming, which enables ultrafast protocols. 

This flexible, modular PCR thermal cycler can connect to other cyclers or computers to increase throughput. 

8. Rotor-Gene Q 6 plex machine ($14,500.00)

The 6 plex machine from the Rotor-Gene Q is one of the real-time PCR machines in the series. The 6 represents the six channels – green, blue, red, orange, crimson, and yellow. The numbers in the other machines correspond to the channels as well. 

The 6-plex machine has top-tier features, including a wide optical range and rotary formats that enhance optical and thermal performance. In addition, the software on the Rotor-Gene Q 6 plex machine is user-friendly, making it very easy to operate.

9. Quidel Sofia 2 ($1,390.00)

Sofia 2 from Quidel is one of the quickest analyzers on the market. The machine delivers SARS-CoV-2 test results within 3 minutes, which is impressive. In this regard, Sofia 2 is a great option for testing multiple samples to detect SARS-CoV-2. 

The features of this molecular diagnostics machine include flexible workflow, customizable settings, and barcode technology. The interface of this analyzer is also very user-friendly, which makes PCR effortless. 

10. Afinion 2 ($2,095.00)

Afinion 2 is another compact analyzer from Abbott. The cartridges of this analyzer are the star of the machine as it is engineered to deliver accurate results. This analyzer also includes self-check systems that work in conjunction with error detection to minimize errors. 

This Abbott analyzer is also one of the systems capable of seamlessly performing multiple analyses simultaneously. The Afinion 2 was originally introduced to test diabetes. However, this FDA-approved analyzer can also run other tests with the same efficiency. 

11. StepOnePlus Real-Time PCR System ($8,999.00)

This real-time PCR system comes with instrumentation and advanced software. In addition, it also has a 4-color optical LED recording system that is highly sensitive. The data analysis of this PCR system is also very intuitive. 

Another great feature of this PCR system is its intuitive, robust software. This real-time PCR system is also ready to use out of the box, making it a perfect machine for first-time users. 

The 96-well system on this real-time PCR delivers quick results. In addition, this setup allows the system to run the data independently of a plate setup or a computer. 

12. Eppendorf Mastercycler ($6,999.00)

We can find several PCR machines from different reputable brands. These machines have different requirements and serve their intended purposes effectively. One such machine currently on the market is the Eppendorf Mastercycler PCR machine. Phenomenal efficiency paired with incredible technology is what the Eppendorf machines are made of. Read to know more about these machines and their uses. 

13. Roche LightCycler 96 Real-Time PCR System ($6,850.00)

The LightCycler 96 from Roche is another compact PCR system that delivers quick results. This thermal cycler has a 96-well system with fiber-optic software that enables data capture and precise thermocycling. 

The gradient functions also facilitate the delivery of accurate COVID-19 test results. In addition, temperature calibration is also straightforward in this thermocycler. Furthermore, the machine delivers quick and accurate results without the need to reference passive dyes. 

The LightCycler 96 has an intuitive touchscreen that enables easy data analysis. Lab technicians can also choose between the connectivity options – a USB stick or a network.

14. Qiagen QIAcube ($2,999.00)

The QIAcube from Qiagen is a classic thermocycler. The spin columns of this machine are quite extraordinary and deliver almost instant results. In this way, the QIAcube eliminates manual work to a large degree. 

QIAcube’s other great feature is its ability to run up to 12 samples per run, accelerating productivity. The touchscreen of this thermocycler is a high-brightness transmissive TFT and is very user-friendly. Besides, the Qiagen software preinstalled in the thermocycler and automated processing also make the entire process effortless. 

Conclusion

The battle with COVID-19 is far from over, and many parts of the globe continue to fight against the virus. PCR thermocyclers have played a pivotal role in detecting the SARS-CoV-2 virus in labs, clinics, hospitals, and even satellite locations. 

Thermal cyclers reduce manual work, expedite testing, and make it convenient to test large batches of COVID-19 samples at once. On top of it all, Best PCR thermal cyclers deliver the most accurate results for detecting SARS-CoV-2, making them the preferred choice for labs and testing agencies worldwide.