CN115725702A - A kind of fluorescent RT-PCR primer and probe, kit and use method of human endogenous RNA internal standard - Google Patents

Abstract

The invention provides a fluorescent RT-PCR primer and probe, a kit and a method of use of human endogenous RNA internal standard. The method of the invention is sensitive and fast, and can be widely used by monitoring the mRNA after human B2M gene transcription. Applied to the quality control of the whole process of sampling, nucleic acid extraction, reverse transcription, PCR, etc. in the detection of pathogenic nucleic acids using RNA as a template, it can provide a new type of human endogenous RNA internal standard selection for fluorescent RT‑PCR detection kits for infectious disease pathogens .

Description

A fluorescent RT-PCR primer and probe, kit and use of human endogenous RNA internal standard method

(1) Technical field

The invention relates to a fluorescent RT-PCR primer and probe for human endogenous RNA internal standard, a kit and a use method thereof.

(2) Background technology

Fluorescent RT-PCR and other gene amplification technologies, as the qualitative and quantitative detection of infectious pathogens, play a vital role in the prevention and control of infectious diseases. As one of the important indicators reflecting the quality of nucleic acid testing, the "internal standard" gene can monitor the whole process of sample sampling, nucleic acid extraction, amplification and other testing links, and can be used as a powerful supplement for positive quality control products.

While using fluorescent RT-PCR technology to detect pathogenic nucleic acids, it can also detect endogenous genes (usually human housekeeping genes) in the sampled samples, which can effectively control the quality of sample collection, nucleic acid extraction and amplification reactions, and avoid false negatives. Increase confidence in results. The new type A H1N1 recommended by the World Health Organization (WHO) in 2009 and the H7N9 avian influenza detection scheme recommended in 2013 all use the human housekeeping internal reference gene RNaseP as the internal standard of fluorescent RT-PCR. , measles, rubella and new crown nucleic acid detection kits have adopted similar strategies.

However, it is worth noting that the previous research of the members of the present invention group found that in the process of fluorescent RT-PCR, the reverse transcriptase of the kit is inactivated or even the reverse transcription step is missing, the internal standard detection of most nucleic acid detection kits is still positive As a result (false positive), all the quality control objectives of setting internal standards cannot be reached, which is a detail worth improving in the current commercially available nucleic acid detection kits. Because RNaseP is used as the internal standard, although the sampling and nucleic acid extraction steps can be effectively monitored, it cannot monitor the important link of reverse transcription amplification in fluorescent RT-PCR, because the nucleic acid after the extraction of the specimen contains a large amount of DNA in the form of RNaseP gene, for example, using GAPDH or β-ACTIN gene as an internal standard has a similar result, and the main problem is that there are areas worthy of improvement in the design of the internal standard primer probe. However, since respiratory infectious diseases such as influenza, measles, rubella and new crown are all RNA viruses, the first step of fluorescent RT-PCR relies on the reverse transcription amplification of reverse transcriptase, which is extremely temperature sensitive and easy to detect. Inactivation during the transportation and storage of the kit, resulting in reagent failure and misjudgment of results, delaying epidemic control.

(3) Contents of the invention

The object of the present invention is to provide a kind of fluorescent RT-PCR specific primer and probe, test kit and detection method for detecting the human endogenous RNA internal standard of B2M gene. 2-Microglobulin, B2M) The primer probe of the intron sequence is used as an internal standard (see Figure 1 for the schematic diagram), and can only be effectively amplified when the B2M gene is transcribed into mRNA (because the intron length is about 4000 bp bases, in the DNA The RNA internal standard will monitor the sampling, reverse transcription, nucleic acid extraction and amplification of fluorescence RT-PCR in the whole process, so as to realize the quality control of the whole process, which can make the experiment The result is more reliable.

The technical scheme adopted in the present invention:

The invention provides a fluorescent RT-PCR specific primer and probe for detecting human endogenous RNA internal standard of B2M gene, the specific primer is a B2M upstream primer and a B2M downstream primer, and the probe is a B2M fluorescent probe , the sequence is as follows:

B2M upstream primer (SEQ ID NO.1): 5'-GGGGGAGGGTTTCTCTTCCGC-3';

B2M downstream primer (SEQ ID NO.2): 5'-GGGCTTTTGCGGGAGCGCATG-3';

B2M fluorescent probe (SEQ ID NO.3):

ROX-AACGCAAATCCCACTGTCACATGCATT-BHQ2; wherein, ROX is a fluorescent reporter group, and BHQ2 is a fluorescent quencher group.

The present invention also provides a kit comprising the specific primers and probes, the kit includes RT-PCR reaction reagents, specific primers, probes, positive controls and negative controls; the positive control is the B2M gene The in vitro transcribed RNA, the negative control is RNase-free H ₂ O.

Preferably, the RT-PCR reaction reagents include: 2×5G qPCR Buffer GB, fluorescent RT-PCR EnzymeMix, DNase/Rnase Free Water.

The present invention also provides a method for detecting the B2M gene with the kit, the method comprising the following steps:

(1) RNA is extracted from the sample to be tested; the sample to be tested includes oropharyngeal swabs, feces, serum, etc.;

(2) Using the sample RNA to be tested as a template, add RT-PCR reaction reagents, specific primers and probes for fluorescent RT-PCR detection; at the same time, perform fluorescent RT-PCR detection for positive controls and negative controls;

(3) Fluorescence RT-PCR detection is based on the Ct value < 37 and the amplification curve is S-shaped as the positive judgment principle; when the Ct value is between 37 and 40, the experiment needs to be repeated, and a good S-shaped amplification curve can be obtained in both experiments. is judged to be positive.

Preferably, the fluorescent RT-PCR detection conditions are: reverse transcription at 52°C for 5 minutes, pre-denaturation at 95°C for 10s, denaturation at 95°C for 5s, then 55°C for 20s, and ROX fluorescence detection at 55°C for a total of 40 cycles.

The key of the present invention lies in the design of specific primers and probes. Other components in the kit are reaction reagents routinely used in the field for fluorescent RT-PCR, such as dNTP mix, MgCl ₂ , buffer, enzymes, etc., which are common in the field. Technicians can choose according to common sense, can prepare by themselves, or can choose commercial products, such as Probe 1-step fluorescent RT-PCR 5G kit (toroivd, QPR-303) kit from TOROIVD company, etc. The reaction system is shown in Table 1.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

1. The specific primers and probes for detecting the B2M gene of the human endogenous RNA internal standard designed by the present invention have good specificity and high sensitivity.

2. The B2M gene is stably expressed in various tissues of the human body, and is suitable for sampling quality monitoring of common samples such as throat swabs, nasal swabs, feces, blood, and sputum.

3. The specific primers and probes of the present invention can monitor the whole process of sampling, nucleic acid extraction, reverse transcription, PCR, etc., and can provide a new type of human endogenous RNA internal standard selection for the fluorescent RT-PCR detection kit of infectious disease pathogens.

(4) Description of drawings

Figure 1. Schematic diagram of the design of B2M gene-specific primers and probes.

Fig. 2. Sensitivity detection results of fluorescent RT-PCR of B2M gene-specific primers and probes; from A to D are 10 ⁴ , 10 ³ , 10 ² , and 10 gene copies.

Figure 3. Test results of human-derived throat swabs, blood and feces and other clinical samples; A to C are 5 throat swabs, 5 stool samples and 3 serum samples in sequence.

(5) Specific implementation methods

The present invention will be further described below in conjunction with specific implementation examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention, and the specific experimental conditions and methods are not indicated in the following implementations.

Example 1: Design of internal standard primers and probes for the specific detection of B2M genes

Find and download the sequence of the B2M gene (NG_012920.2, the nucleotide sequence shown in SEQ ID NO.4) and the corresponding mRNA (NM_004048.3, the nucleotide sequence shown in SEQ ID NO.5) from the NCBI database and Gene map, using DNAMAN and Snapgene software, align and mark the starting position of exons, exon 1 is located at 1-97bp of the mRNA sequence, exon 2 is located at 3907-4185bp, exon 3 is located at 4813-4840bp , exon 4 is located at 6091-6629bp.

Using mRNA as a template, the upstream primer and downstream primer are located on different exons, the probe spans the exon, and the primer probe that spans the intron is designed. The important feature is the length of the distance between the upstream and downstream primers in the DNA state It is about 4000bp, but it is only about 100bp in the state of mRNA. In the state of DNA, the amplification product is too long to be amplified effectively. The design schematic diagram is shown in Figure 1.

B2M upstream primer (SEQ ID NO.1):

5'-GGGGGAGGGTTTCTCTTCCGC-3';

B2M downstream primer (SEQ ID NO.2):

5'-GGGCTTTTGCGGGAGCGCATG-3';

B2M fluorescent probe (SEQ ID NO.3):

ROX-AACGCAAATCCCACTGTCACATGCATT-BHQ2;

Among them, ROX is a fluorescent reporter group, and BHQ2 is a fluorescent quencher group.

Example 2: Sensitivity of fluorescent RT-PCR method for internal standard of human endogenous RNA

(1) Quantify the in vitro transcribed RNA of the B2M gene and make a 10-fold gradient dilution with deionized water to obtain a total of 5 dilutions from 1×10 ⁰ to 1×10 ⁴ copies/μL as a standard template;

(2) The RNA obtained by diluting step (1) is used as a template, and the specific primers and probes of Example 1 are used, and the reaction solution in Table 1 is added to the PCR tube to perform reverse transcription real-time fluorescent quantitative PCR detection; obtain fluorescent amplification The results are shown in Figure 2. Figure 2 shows 10 ⁴ , 10 ³ , 10 ² , and 10 gene copies from A to D in sequence, and the lowest detection limit is determined to be 10 copies/reaction.

The positive control is the in vitro transcribed RNA of the internal reference B2M gene sequence of human RNA; the negative control is RNase-freeH ₂ O.

The amplification reaction conditions were: reverse transcription at 52°C for 5 minutes, pre-denaturation at 95°C for 10s, denaturation at 95°C for 5s, and then 20s at 55°C, and ROX fluorescence detection at 55°C for a total of 40 cycles.

Table 1 is the reagent composition of fluorescent RT-PCR reaction solution (1 person)

Example 3: Detection and verification of clinical samples such as human throat swabs, blood and feces

Randomly select 5 throat swabs, 3 serums, and 5 stool samples stored in the laboratory. The above samples were collected from patients suspected of enterovirus, and informed consent was signed when sampling. After the RNA was extracted from the above specimens, the fluorescent RT-PCR method was used to detect the method in Example 2. The mRNA detection results of B2M could obtain a good amplification curve, and the Ct values of the samples were all less than 37, showing that the RNA detection results of the B2M gene were all Positive (Figure 3).

Claims (7)

1. a fluorescent RT-PCR specific primer and probe for detecting the internal standard of human endogenous RNA of B2M gene, it is characterized in that, described specific primer is B2M upstream primer and B2M downstream primer, and probe is B2M Fluorescent probe, the sequence is as follows: B2M upstream primer: 5'-GGGGGAGGGTTTCTCTTCCGC-3'; B2M downstream primer: 5'-GGGCTTTTGCGGGAGCGCATG-3'; B2M fluorescent probe: ROX-AACGCAAATCCCACTGTCACATGCATT-BHQ2; wherein, ROX is a fluorescent reporter group, and BHQ2 is a fluorescent quencher group. 2. A kit comprising specific primers and probes according to claim 1. 3. test kit as claimed in claim 2, is characterized in that, described test kit comprises RT-PCR reaction reagent, specific primer, probe, positive control and negative control; Described positive control is the in vitro transcription of B2M gene RNA, the negative control is RNase-free H ₂ O. 4. The kit according to claim 2, wherein the RT-PCR reaction reagents include: 2×5G qPCRBuffer GB, fluorescent RT-PCR Enzyme Mix, DNase/Rnase Free Water. 5. a method for the detection of B2M gene by the test kit according to claim 1, is characterized in that, the method comprises the steps: (1) RNA is extracted from the specimen to be tested; (2) Using the sample RNA to be tested as a template, add RT-PCR reaction reagents, specific primers and probes for fluorescent RT-PCR detection; at the same time, perform fluorescent RT-PCR detection for positive controls and negative controls; (3) Fluorescence RT-PCR detection is based on the Ct value < 37 and the amplification curve is S-shaped as the positive judgment principle; when the Ct value is between 37 and 40, the experiment needs to be repeated, and a good S-shaped amplification curve can be obtained in both experiments. is judged to be positive. 6. The method according to claim 5, wherein the fluorescent RT-PCR detection conditions are: reverse transcription at 52°C for 5min, pre-denaturation at 95°C for 10s, denaturation at 95°C for 5s, and then 55°C for 20s, at 55°C ℃ for ROX fluorescence detection, a total of 40 cycles. 7. The method according to claim 5, wherein the samples to be tested include oropharyngeal swabs, feces, and serum.

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