COVID-19 Testing :PCR swab test,antibody test, and antigen test difference.

PCR swab test（Nucleic acid test） and antibody / antigen detection are important means to confirm COVID-19, and also one of the important processes for patients to diagnose. But do you know what their detection principle is? What are the sample types? What is the inspection process and what are the problems? 

COVID-19 nucleic acid test

Nucleic acid detection has the characteristics of early diagnosis, high sensitivity and specificity. At present, real-time fluorescence quantitative RT-PCR is the most widely used technology. PCR swab testing is a very effective way to identify SARS-COV-2.

1. Principle of COVID-19 test kits?

The researchers used sampling swabs to collect samples of the virus, targeting the unique gene sequence of the virus.

By PCR amplification of the virus sample, the target DNA sequence we selected increased exponentially.

Researchers can combine the amplified DNA sequence with pre added fluorescent labeled probe fragments to generate fluorescent signals.

The more target genes amplified, the stronger the cumulative fluorescence signal.

In Virus-free samples, no target gene was amplified. Therefore, researchers can not detect enhanced fluorescence signals.

That is, we do not carry COVID-19.

Therefore, nucleic acid detection is actually to detect the accumulation of fluorescence signal to determine whether there is viral nucleic acid in the sample.

 

2. What are the methods of covid-19 testing samples?

Generally, PCR nasal swab test, pharyngeal swab, nasopharyngeal swab, sputum, bronchial lavage fluid, alveolar lavage fluid, etc.

Sampling swab consists of a nylon flocked swab.what is a flocked swab?

3. Steps of PCR swab test for COVID-19.

At present, pharyngeal swabs are the main sampling method for the diagnosis of novel coronavirus infection. Correct sampling is the key to improve the accuracy of nucleic acid samples collected from pharyngeal swabs. What are the factors affecting the accuracy of nucleic acid samples collected from pharyngeal swabs? There are three main aspects:

Depth of throat swab collection?

How long is the throat swab in contact with the mucosa?

Pharyngeal swab sampling “dot to dot” or wipe?

 

(1) Oral and pharyngeal swab collection tips

1. Sampling position: sit or semi-sit in decuffine position, with head slightly raised and mouth open, and head resting on the wall or against the back of the chair.

2. Standard sampling operation: Sampling personnel press the tongue of the subject with tongue depressors.

Pharyngeal swabs should be performed on both sides of the pharyngeal tonsil with “Quick wipe up and down” and on the posterior pharyngeal wall with “Quick left and right wipe” . Repeat 3 to 4 times.

Avoid contact with tongue, teeth and gums.

If you feel uncomfortable, quit, take a break, and try again.

3. Precautions: Drinking water before oral and pharyngeal swab test will dilute the virus attached to the pharynx, especially drinking hot water will inhibit the activity of the virus.

In order to improve the accuracy of pharyngeal swab nucleic acid detection, we recommend that subjects do not drink water 15-30 minutes before the detection, so as to ensure the high accuracy of pharyngeal swab nucleic acid test.

 

(2) Nasopharyngeal swab collection tips

1.The sampling personnel shall collect samples from the side of the subject, and the subject shall pull down the mask to expose only his nostrils. If there is sneezing reflex, the subject can cover it with elbow or paper towel.

2. Standard sampling operation: “one insertion, two stops and three rotations” — measure the distance from the tip of the nose to the front of the ear, half of the insertion length, usually about 4 cm for adults; Stay for 15-30 seconds to absorb nasopharyngeal secretions (depending on tolerance, minimum stay for at least 3 seconds; Rotate the nasopharyngeal swab for 1 week and slowly remove it.

The above is for the reference of sampling medical staff and patients, let’s work together to defeat COVID-19!

(3)Store samples

Dip the flocked swab head into the cell preservation solution(Virus transport medium), and tighten the tube cap immediately after breaking the tail;

Storage, put the sample tube into a sealed bag for timely inspection, and the inspection process requires a strict transportation environment, 2-8 ℃ storage.

(4) PCR TEST

Researchers operate nucleic acid extraction, extract inactivated virus samples for subsequent nucleic acid detection, and can use automatic equipment such as nucleic acid extractor.

Fluorescence PCR nucleic acid detection, that is, on the machine detection, the extraction of fluorescence PCR amplification reaction, needs 70-80 minutes.

4. What are the problems in nucleic acid testing?

False negative, due to improper sampling, improper specimen preservation, the use of different types of specimens and the use of different manufacturers of reagents may lead to nucleic acid test results “false negative” and missed diagnosis;

■ High requirements for detection equipment or platform, high sensitivity RT-PCR instrument is expensive, laboratory cleanliness and operator requirements are also high;

■ Nucleic acid testing is time-consuming. It usually takes 4-6 hours to complete an RT-PCR test.

Therefore, when the nucleic acid test is negative.

Relevant personnel can increase the detection of IgM and IgG antibodies to make up for the shortcoming that nucleic acid detection is easy to cause missed diagnosis.

 

COVID-19 Testing serum antibody test

Seven days after novel coronavirus pneumonia, the serum specific antibodies gradually emerged. The first is immunoglobulin IgM antibody, and then IgG antibody.

Thus, an elevated IgM antibody indicates a recent acute infection, and an elevated IgG antibody indicates a previous infection.

The biggest advantage of serological testing lies in its convenience and short detection time, which can effectively break through the limitations of existing detection technology on personnel and sites and shorten the detection time.

Novel coronavirus infection can be diagnosed in suspected cases if the serum specific IgM and IgG antibodies are positive, the IgG antibody changes from negative to positive, or the recovery period increases by 4 times or more than the acute period.

1. Principle of serum antibody test kit?

The sample is added to the sample adding hole, and the sample will move forward along the detection card under the action of chromatography.

If the sample contains new coronavirus IgM antibody, the antibody will combine with colloidal gold labeled virus antigen to form a sandwich complex, which will show a positive result.

If the sample contains New Coronavirus IgG antibody, the antibody will bind to colloidal gold labeled New Coronavirus antigen and form a sandwich complex, which will show positive results.

The detection card also contains a quality control line (line C) to judge whether the chromatography process is smooth.

2. What are the sample types for serum antibody detection?

Generally, it is blood, including serum, plasma and whole blood.

3.COVID-19 antibody test in three steps

1. Open the aluminum foil bag of the test card, take out the test card and place it horizontally on the table;

2. Use a pipette to absorb serum/plasma/whole blood sample and add it to the sampling site. Then use the same method to absorb buffer drops and add them to the sampling hole of the test card.

3. Wait 15 minutes and read the result.

4. What are the problems in serum antibody detection?

■ False positive, individual patients with rheumatoid factors in the blood, heterosinophilic antibodies, autoantibodies, drugs and tumor cells, etc., easy to cause cross-reaction test, so occasionally false positive results;

■ False negative, due to the existence of a certain window period of serum antibody detection methods and the sensitivity of the detection kit will appear false negative results.

Therefore, serum antibody test is only used as a supplementary test for suspected cases of novel Coronavirus nucleic acid negative test and cannot be used as a diagnostic indicator for screening alone.

The combined use of serum antibody detection and nucleic acid detection can help to improve the detection rate of the disease and find out the confirmed patients as much as possible, which is more conducive to the control of the epidemic.

Interpretation of results of nucleic acid combined with IgG and IgM antibody detection

COVID-19 Testing antigen detection

A novel coronavirus antigen test can novel coronavirus be detected directly in human body samples. The diagnosis is fast and accurate, with low requirements for equipment and personnel.

1. Principle of antigen detection kit?

SARS-CoV-2 , N, E and S protein antigens stimulate virus cells to produce specific antibodies.

2. What are the types of antigen test samples?

Usually samples from infected sites, e.g. oropharyngeal swabs, nasopharyngeal swabs, sputum, serum, plasma, etc.

 

3.COVID-19 antigen test in four steps

1. Drop the sample treatment liquid into the sample treatment tube;

2. Stir the sample swab and squeeze the tube wall until the specimen is fully eluted into the treatment solution;

3. Take out the detection card and add sample to the round hole;

4. Wait 15 minutes and read the result.

4. What are the problems in antigen detection?

■ False negative, higher sensitivity of antigen detection is required. Because New Coronavirus mainly affects alveolar and other lower respiratory tract. Therefore, sampling from the upper respiratory tract of nasopharynx and oropharynx cannot determine whether the pathogen can be detected. Or the sample contains a small number of viruses. All these factors may cause missed detection.

■ The preparation process is tedious and time-consuming. The development of the antigen kit requires the preparation of the recombinant antigen first, and then the preparation of monoclonal antibody in mice, which takes about two or three months. If the prepared antibody performance is not good, it needs to be prepared again, which takes another two or three months.

The joint detection

The COVID-19 Testing nucleic acid / antibody / antigen has different focuses and can not be substituted for each other. Multiple detection methods are combined and complement each other. Combine molecular biology and immune level detection to give play to their respective advantages and improve sensitivity and specificity. It can effectively shorten the detection window period, improve the positive detection rate, and provide double guarantee for all possible risk groups.

 

reference：Zhengtu Li，Yongxiang Yi et al. Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis. J Med Virol.2020；1-7