The amplification response curve of the novel coronavirus internal standard was serrated because

Author Zhong Guangzhi   The novel coronavirus pneumonia (COVID-19) is a new acute respiratory infectious disease, and has become a major global public health event, Li Aiwen unit, Guangdong second Chinese medicine hospital, said in the introduction of New Coronavirus pneumonia.

More than a year since the outbreak of COVID-19, a variety of mutational strains have attracted special attention.

The most interesting strains include alpha in the UK, beta in South Africa, gamma in Brazil and delta in India.

Delta (delta) strain has now become the most important strain in the global epidemic.

Delta strain has been reported in 132 countries and regions.

Delta strain has also caused local epidemic spread in Guangzhou, Nanjing and other places in China.

The novel coronavirus pneumonia detection plays a key role in the whole staff screening and early detection of novel coronavirus pneumonia cases in the Yellow code and red code personnel investigation and control.

Early detection, early report, early isolation and early treatment can improve the cure rate and reduce the mortality [1].

The inspectors are the main force of the novel coronavirus nucleic acid detection and undertake the heavy task of the novel coronavirus nucleic acid detection.

They should not only strengthen the quality control of the nucleic acid detection, but also ensure the reporting time limit of the nucleic acid detection (report within 6 hours for fever outpatients and emergency patients) [2].

Fluorescence PCR (polymerase chain reaction) is a widely used method for nucleic acid detection in New Coronavirus, which is fast, sensitive and specific.

Because New Coronavirus is a RNA virus, the detection of the kit is basically based on reverse transcription and real-time polymerase chain reaction (RT-PCR), amplifying the nucleic acid (RNA) of the pathogen, and simultaneously detecting the amplified products by fluorescence probe.

At present, the two steps of preparing reagent and adding template in RT-PCR detection in most PCR laboratories are still manual, which will inevitably lead to artificial operation errors, resulting in false negative or false positive results.

To solve this problem novel coronavirus laboratory testing expert consensus [3] gave the following countermeasures: in addition to quality control of reagents and operations, control should be set up: in addition to at least 3 negative controls, 1 weak positive controls (third quality control products) and internal standard control holes, each batch should be set up blank control to monitor laboratory pollution.

However, it did not explain whether the abnormal internal standard amplification curve needs to be rechecked.

The following are two cases of abnormal internal standard amplification curve encountered by the author during the novel coronavirus nucleic acid test on duty.

The specific reports are as follows.

Recently, the author encountered an abnormal PCR amplification response curve on the night shift – the internal standard amplification response curve was serrated, which was reminiscent of the ECG of ventricular fibrillation.

In the quiet PCR room in the early morning, I felt my heart trembling and my heart racing » racing » racing » racing ».

When continuing to check the amplification curves of other samples, it was found that there was no amplification curve in the internal standard of sample 2018, as shown in Figure 1.

  Fig.

1 amplification response curve of abnormal internal standard (a) shows that the internal standard of sample 2011 shows a zigzag curve; (B) It is the internal standard of sample 2018, and there is no amplification curve; (C) It is the normal internal standard amplification curve of sample 2010.

In order to find out the reason, I checked the amplification reaction curves of negative control, positive control and positive quality control in turn.

The results are shown in Figure 2.

Fig.

2 control amplification reaction curve (a) is the negative control; (B) Positive control; (C) It is positive quality control.

Then check the amplification response curves of three normal saline controls and reagent blank controls, as shown in Figure 3.

The results showed no abnormality.

Fig.

3 control amplification response curve (a) is normal saline control 1; (B) Normal saline control 2; (C) Normal saline control 3; (D) It is the reagent blank control.

Case analysis question 1: why is the internal standard of sample 2011 serrated amplification response curve? Question 2: why is there no amplification curve in the internal standard of sample 2018? For doubt 2 is relatively simple, because we often encounter this situation, “New Coronavirus laboratory testing experts consensus” [3] pointed out that the false negative results are mainly caused by: (1) at different times of infection, there are differences in viral load in different parts of the body.

(2) Sample collection is not standardized; (3) Improper specimen transport, preservation or inactivation methods lead to RNA degradation; (4) The virus gene sequence changed.

In addition, there are human operation factors, such as not adding the template or adding the template to other holes (wrong holes) or not adding reagents, which will also cause false negative results of nucleic acid detection.

But I’m not a novice anymore.

I can’t make these low-level mistakes.

Because there is no time and thought to ensure the report time, recheck first according to the recheck process: 1.

Recheck of the original extraction plate template + re extraction plate template: the nucleic acid was extracted from the samples No.

2011 and 2018 again, and the original extraction plate template and the new extraction plate template were rechecked at the same time.

The results are shown in Figure 4.

The results showed that the template internal standard of samples 2011 and 2018, whether the original plate or re extracted plate, had amplification curves, and the nucleic acid test result was negative.

Fig.

4 PCR amplification reaction curve of original plate and re extraction template (a) shows the results of original plate No.

2011; (B) Re extraction results for 2011; (C) Is the result of original plate No.

2018; (D) Re extract the results for 2018.

At the same time, check the results of negative control, positive control, positive quality control and normal saline control, as shown in Figure 5.

The results show that there is no problem in excluding the factors of sampling and the first extracted nucleic acid and RNA.

However, the internal standard reaction curve of normal saline has tail warping phenomenon, because the reagent adopts endogenous internal standard, which is labeled with human gene.

This gene exists in human epidermal cells, and human pollution may occur during operation.

In addition, environmental pollution may also lead to the growth of internal standard.

It is suggested that we pay attention to the pollution of operators in the future, Change gloves frequently and conduct environmental sample quality control to monitor environmental pollution.

Fig.

5 the control amplification response curve (a) of the retested sample is the negative control; (B) Positive control; (C) Positive quality control; (D) It is normal saline control.

two   Recheck of high sensitivity rapid detector: in order to ensure the reporting time limit, the samples No.

2011 and No.

2018 were rapidly tested with a more sensitive instrument with a detection limit of 200 copies / ml, and the results are shown in Fig.

6.

The results showed that the internal standard of samples 2011 and 2018 had amplification curves, and the nucleic acid test result was negative.

Fig.

6 constant temperature amplification reaction curve (a) shows sample 2011; (B) It is specimen 2018.

So what is the reason why there is no amplification curve for the first internal standard of specimen 2018? The re inspection results can eliminate the causes of sample collection and extraction problems, and there will be no errors and omissions after the reagent is confirmed.

The most likely reason is that the operation adds the wrong hole.

Long time busy and high mental tension are easy to make people tired, but you have to concentrate on adding samples to the dense small holes, which will inevitably lead to small errors.

Since the outbreak of COVID-19, night shift nucleic acid testing has tested our physical strength, and on the other hand, it has exercised our emergency response ability.

The internal standard has no amplification or tail warping ➯ recheck, and everyone is basically familiar with the road.

It was the first time that the internal standard of sample 2011 was sawtooth amplification, and the reaction curve was met for the first time.

After the shift was handed over the next morning, I asked other colleagues that I had not encountered this situation.

Later, I asked senior brother Li, who was experienced.

He explained to me that there were small bubbles in the reaction tube.

When the temperature increased during the extension process, the small bubbles increased and burst, resulting in a sudden decrease in the fluorescence value detected by the instrument, Repeated bubble rupture leads to the appearance of sawtooth curve..