COVID-19 RT-PCR

COVID-19 RT-PCR

About RT-PCR:

The COVID-19 RT-PCR Test is a real-time reverse transcription polymerase chain reaction (rRT -PCR) test. When multiplexed into a single reaction, the test uses two primers and probe sets to detect two regions in the SARS-CoV-2 N gene and one primer and probe set to detect RP.

Human Coronavirus Types
Coronaviruses are named for the crown-like spikes on their surface. There are four main sub groupings of coronaviruses, known as alpha, beta, gamma, and delta. Human coronaviruses were first identified in the mid-1960s. The seven coronaviruses that can infect people are:

Common human coronaviruses

1)      229E (alpha coronavirus)

2)      NL63 (alpha coronavirus)

3)      OC43 (beta coronavirus)

4)      HKU1 (beta coronavirus)

Other human coronaviruses

5)      MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS)

6)      SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS)

7)      SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19)

People around the world commonly get infected with human coronaviruses 229E, NL63, OC43, and HKU1.

Sometimes coronaviruses that infect animals can evolve and make people sick and become a new human coronavirus. Three recent examples of this are 2019-nCoV, SARS-CoV, and MERS-CoV

How is the COVID-19 Virus Detected using Real Time RT-PCR?

What is real time RT-PCR?

Real time RT-PCR is a nuclear-derived method for detecting the presence of specific genetic material from any pathogen, including a virus. Originally, the method used radioactive isotope markers to detect targeted genetic materials, but subsequent refining has led to the replacement of the isotopic labelling with special markers, most frequently fluorescent dyes. With this technique, scientists can see the results almost immediately while the process is still ongoing; conventional RT-PCR only provides results at the end. 

While real time RT-PCR is now the most widely used method for detecting coronaviruses, many countries still need support in setting up and using the technique. 

What is a virus? What is genetic material?

A virus is a microscopic package of genetic material surrounded by a molecular envelope. The genetic material can be either DNA or RNA. 

DNA is a two-strand molecule that is found in all organisms, such as animals, plants, and viruses, and it holds the genetic code, or blueprint, for how these organisms are made and develop. 

RNA is generally a one-strand molecule that copies, transcribes and transmits parts of the genetic code to proteins so they can synthetize and carry out functions that keep organisms alive and developing. There are different variations of RNA that do the copying, transcribing and transmitting. 

Some viruses such as the coronavirus (SARS-Cov2) only contain RNA, which means they rely on infiltrating healthy cells to multiply and survive. Once inside the cell, the virus uses its own genetic code — RNA in the case of the coronavirus — to take control of and ‘reprogramme’ the cells so that they become virus-making factories.   

In order for a virus like the coronavirus to be detected early in the body using real time RT-PCR, scientists need to convert the RNA to DNA. This is a process called ‘reverse transcription’. They do this because only DNA can be copied — or amplified — which is a key part of the real time RT-PCR process for detecting viruses. 

Scientists amplify a specific part of the transcribed viral DNA hundreds of thousands of times. Amplification is important so that instead of trying to spot a minuscule amount of the virus among millions of strands of genetic information, scientists have a large enough quantity of the target sections of viral DNA to accurately confirm that the virus is present.

How does real time RT-PCR work with the coronavirus?

A sample is collected from parts of the body where the coronavirus gathers, such as a person’s nose or throat. The sample is treated with several chemical solutions that remove substances, such as proteins and fats, and extracts only the RNA present in the sample. This extracted RNA is a mix of a person’s own genetic material and, if present, the coronavirus’ RNA. 

The RNA is reverse transcribed to DNA using a specific enzyme. Scientists then add additional short fragments of DNA that are complementary to specific parts of the transcribed viral DNA. These fragments attach themselves to target sections of the viral DNA if the virus is present in a sample. Some of the added genetic fragments are for building DNA strands during amplification, while the others are for building the DNA and adding marker labels to the strands, which are then used to detect the virus. 

The mixture is then placed in a RT-PCR machine. The machine cycles through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target sections of viral DNA. The cycle repeats over and over to continue copying the target sections of viral DNA. Each cycle doubles the previous amount: two copies become four, four copies become eight, and so on. 

A standard real time RT-PCR setup usually goes through 35 cycles, which means that by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample. 

As new copies of the viral DNA sections are built, the marker labels attach to the DNA strands and then release a fluorescent dye, which is measured by the machine’s computer and presented in real time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. When the amount goes over a certain level of fluorescence, this confirms that the virus is present. Scientists also monitor how many cycles it takes to reach this level in order to estimate the severity of the infection: the fewer the cycles, the more severe the viral infection is. 

Why use real time RT-PCR?

The real time RT-PCR technique is highly sensitive and specific and can deliver a reliable diagnosis as fast as three hours, though usually laboratories take on average between 6 to 8 hours. Compared to other available virus isolation methods, real time RT-PCR is significantly faster and has a lower potential for contamination or errors as the entire process can be done within a closed tube. It continues to be the most accurate method available for detection of the coronavirus. 

To detect past infections, which is important for understanding the development and spread of the virus, real time RT-PCR cannot be used as viruses are only present in the body for a specific window of time. Other methods are necessary to detect, track and study past infections, particularly those that may have developed and spread without symptoms.

1. Specimen collection:

Specimen Type and Priority:

• A nasopharyngeal (NP) specimen
• An oropharyngeal (OP) specimen
• A nasal mid-turbinate swab specimen
• An anterior nares (nasal swab) specimen
•  Nasopharyngeal wash/aspirate or nasal wash/aspirate (NW) specimen collected by a healthcare provider.

2019-NOVEL CORONAVIRUS (COVID-19) SPECIMEN COLLECTION KIT INSTRUCTIONS

Use: To collect nasopharyngeal specimens for 2019-Novel Coronavirus (SARS-CoV-2), the virus that causes COVID-19. 

Kit contents: 1 Tube of Universal Transport Media (UTM)
1 Nasopharyngeal swab (smaller swab, flexible shaft) (CDC recommended)
1 Ziploc specimen bag containing absorbent pad
1 Laboratories Test Requisition form
1 Ice pack (keep in a freezer until ready to package & transport specimens)Label tube of UTM legibly with the patient’s name and date of birth, or medical record number (specimen tube must have two unique patient identifiers on it, or it will not be tested).

Nasopharyngeal specimen: (CDC recommended specimen type)
    • Use the flexible shaft NP swab provided to collect the specimen.
    • Have the patient blow their nose and then check for obstructions.
    • Tilt the patient’s head back 70 degrees & insert the swab into nostril parallel to the palate (not upwards) until resistance is encountered or the distance is equivalent to that from nostrils to outer opening of patient’s ear indicating contact with nasopharynx. Leave swab in place for several seconds to absorb secretions. Slowly remove the swab while rotating it.

• Insert the swab into the tube of UTM, making certain that the swab tip is covered by the liquid in the tube. The swab is to remain in the tube for transport. 
• Plastic shaft NP swab: The swab shaft extends past the top of the tube. Snap off at the brake line on the shaft, allowing the end with the swab tip to remain in the liquid. The tip of the swab must be immersed in the liquid. 
• Wire shaft swab: cut the upper end of the wire with clean scissors so that it is even or below the top of the vial, allowing the end with the swab tip to remain in the liquid.

• Throat specimen (Oropharyngeal swab [OP]):

If nasopharyngeal specimen can not be collected due to inability to procure np swabs, a throat swab can be sent as an alternative specimen.

NOTE: Throat swab tips must be synthetic (ex: polyester, rayon, or dacron). Cotton or calcium alginate tipped, or wooden shaft swabs are unacceptable

• Use a throat swab to collect specimen by swabbing the patient’s posterior pharynx and tonsillar area (avoid the tongue).

• Insert the swab into the vial of UTM. If the swab shaft extends past the top of the tube, clip it so that the top of the swab shaft it is just below the top of the tube allowing the end with the swab tip to remain in the liquid. The swab tip must be immersed in the liquid.

Next Steps:

• Securely tighten the cap on the tube of UTM and recheck to make certain it is labeled with two patient identifiers. Write “NP” or “OP” on the tube of UTM. Insert tube into specimen transport bag and close bag tightly.

• Complete a Laboratories test requisition form. For test requested, write “COVID-19" under Comments/Other test requests. Ensure that all information is legible, complete and accurate. Place the completed form into the outside pocket of the specimen bag. Do not enclose it inside the bag with the specimen tube.

Holding:

Store specimens refrigerated (2 – 8 °C) until ready to send to the laboratory. Specimens may be held refrigerated for up to 72 hours. Specimens must be received at the Laboratories within three days of being collected.

Packaging:

Specimens must be packaged and shipped in accordance with appropriate Department of Transportation (DOT) regulations for Category B Biological Substances.

• Place the securely sealed Ziploc bag containing the specimen tube along with the frozen ice pack into the styrofoam box that is contained within the cardboard shipping box. If using an alternate type of packaging, make certain it is compliant and labeled appropriately for Category B Biological Substances.

• Ensure that the outer packing box that is supplied with the specimen kit is sealed

securely with packaging tape.

• Ensure shipper label information for your facility is completed in full and affixed

to the outer package.

Transport: Submitters are responsible for arranging for specimens to be transported to the Laboratory. 

2. Follow the test manual according to the test procedure insert kit. 

Special COVID-19 ECHO session

Total processing time 6 to 8 hours by semi-automatic RT-PCR machine.

Molecular Assays for Detection of Viral Nucleic Acids:

SARS-CoV-2 is a single-stranded, positive-sense RNA virus, and since its entire genetic sequence was uploaded to the Global Initiative on Sharing All Influenza Data (GISAID) platform on January 10, 2020, companies and research groups in a matter of weeks have developed a range of diagnostic kits for COVID-19. 

The availability of sequence data has facilitated the design of primers and probes needed for the development of SARS-CoV-2-specific testing. 

Reverse Transcription-Polymerase Chain Reaction (RT-PCR):

RT-PCR relies on its ability to amplify a tiny amount of viral genetic material in a sample and is considered to be the gold standard for the identification of the SARS-CoV-2 virus. Currently, RT-PCR tests for COVID-19 generally use samples collected from the upper respiratory system using swabs. In addition, a few studies have also been done using the serum, stool, or ocular secretions. 

RT-PCR starts with the laboratory conversion of viral genomic RNA into DNA by RNA-dependent DNA polymerase (reverse transcriptase). This reaction relies on small DNA sequence primers designed to specifically recognize complementary sequences on the RNA viral genome and the reverse transcriptase to generate a short complementary DNA copy (cDNA) of the viral RNA. 

In real-time RT-PCR, the amplification of DNA is monitored in real-time as the PCR reaction progresses. This is done using a fluorescent dye or a sequence-specific DNA probe labeled with a fluorescent molecule and a quencher molecule, as in the case of TaqMan assays. An automated system then repeats the amplification process for about 40 cycles until the viral cDNA can be detected, usually by a fluorescent or electrical signals.

Reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR creates a cDNA copy of a specific segment of the viral RNA, which is converted to dsDNA that is exponentially amplified

RT-PCR has traditionally been carried out as a one-step or a two-step procedure. One-step real-time RT-PCR uses a single tube containing the necessary primers to run the entire RT-PCR reaction. Two-step real-time RT-PCR involves more than one tube to run the separate reverse transcription and amplification reactions but offers greater flexibility and higher sensitivity than the one-step procedure. 

It requires less starting material and allows for the ability to stock cDNA for the quantification of multiple targets. The one-step procedure is generally the preferred approach for the detection of SARS-CoV-2 because it is quick to set up and involves limited sample handling and reduced bench time, decreasing chances for pipetting errors and cross-contamination between the RT and real-time PCR steps. 

To date, the majority of molecular diagnostic tests have utilized the real-time RT-PCR technology targeting different SARS-CoV-2 genomic regions, including the ORF1b or ORF8 regions, and the nucleocapsid (N), spike (S) protein, RNA-dependent RNA polymerase (RdRP), or envelope (E) genes.

Isothermal Nucleic Acid Amplification: 

RT-PCR requires multiple temperature changes for each cycle, involving sophisticated thermal cycling equipment. Isothermal nucleic acid amplification is an alternative strategy that allows amplification at a constant temperature and eliminates the need for a thermal cycler. Therefore, several methods based on this principle have been developed.

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP):

RT-LAMP has been developed as a rapid and cost-effective testing alternative for SARS-CoV-2. RT-LAMP requires a set of four primers specific for the target gene/region to enhance the sensitivity and combines LAMP with a reverse transcription step to allow for the detection of RNA.

The amplification product can be detected via photometry, measuring the turbidity caused by magnesium pyrophosphate precipitate in solution as a byproduct of amplification. The reaction can be followed in real-time either by measuring the turbidity or by fluorescence using intercalating dyes. Since real-time RT-LAMP diagnostic testing requires only heating and visual inspection, its simplicity and sensitivity make it a promising candidate for virus detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP):

• Step 1: At the 3′-end of the viral RNA, reverse transcriptase and BIP primer initiate conversion of RNA to cDNA.
• Step 2: At the same end, DNA polymerase and B3 primer continue to generate the second cDNA strand to displace and release the first cDNA strand.
• Step 3: The FIP primer binds to the released cDNA strand and DNA polymerase generates the complementary strand.
• Step 4: F3 primer binds to the 3′ end, and DNA polymerase then generates a new strand while displacing the old strand. LAMP cycling produces various sized double-stranded looped DNA structures containing alternately inverted repeats of the target sequence as detected by a DNA indicator dye. Reagents* Primers and master mix containing reverse transcriptase, DNA polymerase with strand displacement activity, dNTPs, and buffers.

Transcription-Mediated Amplification (TMA):

TMA is a patented single tube, isothermal amplification technology modeled after retroviral replication which can be used to amplify specific regions of either RNA or DNA much more efficiently than RT-PCR. It uses a retroviral reverse transcriptase and T7 RNA polymerase and has been used for the detection of nucleic acids from multiple pathogens.

On the basis of this principle, Hologic’s Panther Fusion platform has the capability to perform both RT-PCR and TMA. The Panther fusion platform is distinctive because of its high testing throughput (up to 1000 tests in 24 h) and its capability to simultaneously screen for other common respiratory viruses whose symptoms overlap with COVID-19 using the same patient sample and collection vial.

The initial step involves hybridization of the viral RNA target to a specific capture probe and an additional oligonucleotide containing a T7 promoter primer, which is captured onto magnetic microparticles by application of a magnetic field. Then, the captured RNA target hybridized to the T7 promoter primer is reverse transcribed into a complementary cDNA. 

The RNase H activity of the reverse transcriptase subsequently degrades the target RNA strand from the hybrid RNA–cDNA duplex, leaving a single-stranded cDNA, which includes the T7 promoter. An additional primer is used to generate a double-stranded DNA, which is subsequently transcribed into RNA amplicons by T7 RNA polymerase. 

These new RNA amplicons then reenter the TMA process allowing this exponential amplification process to generate billions of RNA amplicons in less than 1 h. The detection process involves the use of single-stranded nucleic acid torches that hybridize specifically to the RNA amplicon in real-time. Each torch is conjugated to a fluorophore and a quencher. When the torch hybridizes to the RNA amplicon, the fluorophore is able to emit a signal upon excitation.

CRISPR-Based Assays: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) represents a family of nucleic acid sequences found in prokaryotic organisms, such as bacteria. These sequences can be recognized and cut by a set of bacterial enzymes, called CRISPR-associated enzymes, exemplified by Cas9, Cas12, and Cas13. Certain enzymes in the Cas12 and Cas13 families can be programmed to target and cut viral RNA sequences.

Two companies, Mammoth Biosciences and Sherlock Biosciences, established by the CRISPR pioneer scientists, are independently exploring the possibility of using the gene-editing CRISPR methodology for detection of SARS-CoV-2. The SHERLOCK method developed by Sherlock Biosciences uses Cas13 that is capable of exciting reporter RNA sequences in response to activation by SARS-CoV-2-specific guide RNA.

The DETECTOR assay by Mammoth Biosciences relies on the cleavage of reporter RNA by Cas12a to specifically detect viral RNA sequences of the E and N genes, followed by isothermal amplification of the target, resulting in a visual readout with a fluorophore. 

These CRISPR-based methods, as depicted in Figure 3, do not require complex instrumentation and can be read using paper strips to detect the presence of the SARS-CoV-2 virus without loss of sensitivity or specificity. These tests are both low-cost and can be performed in as little as 1 h. These tests have great potential for point-of-care diagnosis.

Two alternative CRISPR methods for detecting viral RNA. Method A (SHERLOCK assay: RT-RPA (recombinase polymerase amplification) converts viral RNA to dsDNA. T7 transcription generates complementary RNA from the dsDNA template. The Cas13–tracrRNA complex binds to the target sequence, which activates the general nuclease enzyme activity of Cas13 to cleave the target sequence and the fluorescent RNA reporter.

Method B (DETECTOR assay: RT-RPA (recombinase polymerase amplification) converts viral RNA to dsDNA. The Cas12a–tracrRNA complex binds to the target sequence, which activates the general nuclease enzyme activity of Cas12a to cleave the target sequence and the fluorescent RNA reporter.

Rolling Circle Amplification:

An alternative method of isothermal nucleic acid amplification known as rolling circle amplification (RCA) has attracted considerable attention for nucleic acid detection since in isothermal conditions, it is capable of 109-fold signal amplification of each circle within 90 min. 

RCA is advantageous in that it can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results frequently encountered in PCR-based assays. An efficient assay for the detection of SARS-CoV by RCA was previously performed in both liquid and solid phases and used to test clinical respiratory specimens. This method, however, has not been deployed for the detection of SARS-CoV-2 at this point. 

Nucleic Acid Hybridization Using Microarray:

Microarray assays have been used for rapid high-throughput detection of SARS-CoV nucleic acids.They rely on the generation of cDNA from viral RNA using reverse transcription and subsequent labeling of cDNA with specific probes. The labeled cDNAs are loaded into the wells of microarray trays containing solid-phase oligonucleotides fixed onto their surfaces. 

If they hybridize, they will remain bound after washing away the unbound DNA, thus signaling the presence of virus-specific nucleic acid. The microarray assay has proven useful in identifying mutations associated with SARS-CoV and has been used to detect up to 24 single nucleotide polymorphisms (SNP) associated with mutations in the spike (S) gene of SARS-CoV with 100% accuracy.

Nucleic acid hybridization using microarray:

Viral cDNA and reference cDNA with different fluorescent labels are mixed and applied to the microarray wells coated with specific DNA probes.

The ability to detect different emergent strains of SARS-CoV-2 may become necessary as the COVID-19 pandemic evolves, and microarray assays provide a platform for rapid detection of those strains as a result of the mutational variation. Although one of the drawbacks of microarray testing has been the high cost generally associated with it, a nonfluorescent, low-cost, low-density oligonucleotide array test has been developed to detect multiple coronavirus strains with sensitivity equal to that of individual real-time RT-PCR. 

In addition, a portable diagnostic platform based on the microarray chip has been used to identify nucleic acids specific to the MERS coronavirus as well as to influenza and respiratory syncytial viruses.

3. Results:

If you test positive for COVID-19 by a viral test, know what protective steps to take if you are sick or caring for someone.

If you test negative for COVID-19 by a viral test, you probably were not infected at the time your sample was collected. However, that does not mean you will not get sick. The test result only means that you did not have COVID-19 at the time of testing.

 If you test positive or negative for COVID-19, no matter the type of test, you still should take preventive measures to protect yourself and others. 

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