Common problems and solutions of nucleic acid detection in PCR laboratory, common abnormal curve summary!

In the past three years, the daily inspection work must be inseparable from nucleic acid detection. After all, the inspectors are the main force of nucleic acid detection. We should not only ensure the effectiveness of the detection results, but also face the risk of infection at all times. This article explains the "Precautions for the Operation of New Corona Nucleic Acid Detection in the PCR Laboratory", focusing on the physiological characteristics of the new coronavirus, the principle of fluorescent PCR, the preparation specification of the PCR laboratory, the operation of the sample detection link, the laboratory data analysis, and the analysis of common problems.

1、 Infectious source

At present, the main source of infection is the patients infected by the new coronavirus. Asymptomatic infections may also become a source of infection.

2、 Route of transmission

The main route of transmission is through respiratory droplets and close contact. It is possible to propagate through aerosols when exposed to high concentration aerosols for a long time in a relatively closed environment. Since a new type of coronavirus can be isolated from feces and urine, it should be noted that feces and urine cause aerosol or contact transmission to environmental pollution.

3、 Principle of Fluorescent PCR

Repeat the three processes of "denaturing annealing extension", and the amount of template DNA will increase exponentially. Theoretically, after n cycles, the amount of template will reach the nth power of 2.

4、 FAQ analysis

① How to deal with the tail lifting of the new crown reagent?

A: If it is a single channel with tail sticking, whether it is FAM or VIC channel, first recheck according to the requirements and re sampling is recommended. If it is not convenient to re sample, it is recommended to re extract the nucleic acid from the original sample for verification. If the recheck is negative and the recheck is still the same channel with tail sticking, carefully review the results. First, eliminate laboratory pollution and second, understand the patient information clearly, According to the clinical symptoms and contact history, if it cannot be confirmed, it is recommended to re sample and recheck. If necessary, another reagent can be used for verification. If there are a large number of weak positive amplification tails, the primary reason is to consider the laboratory pollution, which can be eliminated by making environmental samples and negative controls for verification.

② Are N gene and 1ab gene unique to new coronavirus, and which is more sensitive? Does it overlap with other coronavirus fragments?

A: Both N gene and 1ab are specific and will not cross react with other respiratory pathogens. When designing, the sequences of the two genes are different, resulting in different sensitivity. The sensitivity of the N gene is higher than that of the 1ab gene.

③ How to interpret the inconformity between the test results of the covid-19 and the clinical diagnosis?

A: The new coronavirus is RNA virus and easy to degrade. In addition, the detection results are also related to sampling, nucleic acid extraction and the nucleic acid concentration of the sample itself. It needs to be analyzed one by one and can be interpreted in combination with clinical symptoms.

④ If normal saline is used as the negative control, the internal standard will also increase. How to explain this?

Answer: Because the reagent uses an endogenous internal standard, which is marked by a human gene. This gene exists in human epidermal cells, and human pollution may occur during the operation. In addition, environmental pollution may also lead to the growth of internal standards. This is also the previous suggestion for environmental sample quality control.

⑤ How to deal with the failure of internal standard of covid-19 sample?

A: If the internal standard of the covid-19 sample cannot be found, the sample must be rechecked, and the sample can be remixed to extract nucleic acid. If the internal standard of the sample cannot be found again, it means that the sampling is unqualified, and the clinical department should be notified to re sample and recheck if the extraction problem is ruled out.

⑥ When collecting environmental samples to detect new coronavirus, why can't most samples have internal standards, and occasionally individual samples have internal standards?

Answer: Because the covid-19 reagent detects the human endogenous internal standard, the environmental samples generally do not contain human cells, so the internal standard cannot be detected; The internal standard is occasionally detected. It is possible that there is human derived cell adhesion on the environmental sample, so the internal standard is detected.

In addition to the common problems of nucleic acid detection that inspectors often encounter, the monitor also organized the analysis of common abnormal results of fluorescent PCR today.

Fluorescent PCR has become a conventional detection technology to detect pathogens. In the process of work, we have also encountered various and strange problems. Now we will share with you some of the abnormalities of PCR amplification curve and some experience we have encountered in our laboratory.

1. First, let's look at the normal PCR amplification curve, which is a relatively smooth S-shaped curve, as shown in the figure below:

2. Analysis of common abnormal results

2.1 Case 1

2.1.1 Phenomenon: common single gene tail lifting with large Ct value and single gene tail lifting without Ct value, as shown in the following figure:

2.1.2 Common causes: the negative QC curve is cocked, and there is positive QC or positive sample contamination; The tail of the sample curve indicates that the target gene concentration is low or there is non-specific amplification or contamination.

2.1.3 Solution: first, eliminate whether there is pollution; After eliminating the cause of contamination, retest the sample. If the curve after retesting is a negative straight line, it means that the previous tail lifting is non-specific amplification. If the curve after retesting is still consistent with the previous one, it means that the concentration of nucleic acid of the virus to be tested in the sample is low.

2.2 Case 2

2.2.1 Phenomenon: After the extracted nucleic acid template is added to the PCR reaction system, the PCR tube cap needs to be covered. Due to the large number of samples every day, it is inevitable that the tube cap is not tightly covered. After the completion of PCR, the liquid in the reaction system decreased (see Figure 2c below), and the amplification curve of each fluorescent channel appeared to be oblique linear (see Figure 2a, 2b below), which caused laboratory pollution in serious cases.

2.2.2 Common causes: the pipe cover is not tightly covered, the reaction solution evaporates, the volume in the pipe decreases, and the standard for reading fluorescence changes, resulting in the curve rising in a oblique straight line.

2.2.3 Solution: During operation, pay attention to tightly cover the pipe cover, and use qualified consumables matching the pipe and cover.

2.3 Case 3

Similar to the S-shaped curve, but not a smooth typical S-shaped curve, which is obviously different from the positive QC, as shown in the following figure:

2.3.2 Common causes: there may be interfering substances in the sample, incomplete extraction and purification, and abnormal hot cap of PCR instrument.

2.3.3 Solution: retest; Re extract, or use a different kit to extract.

2.4 Case 4

2.4.1 Phenomenon: irregular amplification curve (see the figure below):

2.4.2 Common causes: There is no abnormality in other hole position curves of the whole board. It is possible that the solution at the hole position is affected by bubbles, which can refract the optical path. When the bubbles break, it will cause a sharp change in the fluorescent signal and interfere with the instrument's collection of the fluorescent signal.

2.4.3 Solution: ① Reduce the generation of bubbles when adding samples; ② Before getting on the machine, snap off the bubbles and centrifuge.

2.5 Case 5

2.5.1 Phenomenon: abnormal endogenous internal parameter curve.

2.5.2 Common cause: foreign matters in the hole; Abnormal temperature or fluorescence signal acquisition; Inhibitors exist in the reaction system; Failed to extract nucleic acid; Unqualified sample collection; The sample/nucleic acid was omitted.

2.5.3 Solution: Open the cover of the PCR instrument to check whether there is any foreign matter in the hole. If there is any foreign matter, use forceps to clamp it out, or use ear ball to blow it out; Analyze whether the internal standard of the hole has been amplified in the previous batch of results. If the internal standard has not been amplified for two consecutive times, consider that the temperature of the hole is abnormal or the fluorescence signal acquisition is abnormal, and calibration or repair is required. After the above conditions are excluded, the original specimen shall be retested, and a report can be issued normally if the internal standard is normal; If the internal standard is still not amplified, re sampling and re inspection are required.

2.6 Case 6

2.6.1 Phenomenon: abnormal external internal standard curve

The uniformity of the external internal standard is good (for example, 12 holes in row G have internal standard curves, and their CT values are uniform, see the figure below)

However, it is sometimes found that the internal standards in the same row or row are relatively discrete, and even multiple internal standards cannot be raised (for example, F7, F9, F10, F11 holes in row F have no internal standard curve, no CT value, and CT values of other holes are relatively discrete, as shown in the figure below)

2.6.2 Common causes: ① The discharge gun and the gun head do not match, and the combination is not tight, resulting in insufficient/uneven sample dosage; ② When the internal standard mixed liquid is sucked by the row gun, the liquid blocks part of the gun head filter element, resulting in uneven sample amount when the sample is added to the cracking plate, or even no liquid is added to some holes.

2.6.3 Solution: It is recommended to use the gun head with matching range, and the pipette should slowly suck; Carefully observe whether the liquid is pumped out during reagent subpackage and sample adding.