CN115725702A - Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer and probe for human endogenous RNA (ribonucleic acid) internal standard, kit and using method - Google Patents

Abstract

The invention provides a fluorescent RT-PCR primer and probe of human endogenous RNA internal standard, a kit and a using method thereof, the method is sensitive and rapid, mRNA after transcription of human B2M gene can be monitored, the method can be widely applied to full-process quality control of sampling, nucleic acid extraction, reverse transcription, PCR and the like in pathogen nucleic acid detection with RNA as a template, and a novel human endogenous RNA internal standard selection can be provided for infectious disease pathogen fluorescent RT-PCR detection kits.

Description

Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer and probe for human endogenous RNA (ribonucleic acid) internal standard, kit and using method

(I) the technical field

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

(II) background of the invention

The gene amplification technology such as fluorescence RT-PCR and the like plays an important role in epidemic prevention and control of infectious diseases as qualitative and quantitative detection of infectious disease pathogens. The internal standard gene is used as one of important indexes for embodying the detection quality of nucleic acid, can carry out full-flow monitoring on detection links such as sample sampling, nucleic acid extraction, amplification and the like, and can be used as powerful supplement of positive quality control products.

The fluorescent RT-PCR technology is utilized to detect pathogenic nucleic acid, and simultaneously, endogenous genes (usually human housekeeping genes) in the collected sample are detected, so that the quality control can be effectively carried out on the processes of sample collection, nucleic acid extraction, amplification reaction and the like, false negative is avoided, and the reliability of the result is improved. The World Health Organization (WHO) recommends a novel H1N1 type in 2009 and a H7N9 avian influenza detection scheme in 2013 both adopt a human housekeeping reference gene RNase P as an internal standard of fluorescent RT-PCR, and similar strategies are adopted in influenza, measles, rubella and neocoronal nucleic acid detection kits approved by the national drug administration at present.

However, it is worth noting that the previous research of the group members of the present invention found that, in the case of inactivation of reverse transcriptase of the kit during the fluorescent RT-PCR process or even the absence of the reverse transcription step, the internal standard detection of most nucleic acid detection kits still has a positive result (false positive), and does not reach all quality control targets of the internal standard, which is one of the details of the currently marketed nucleic acid detection kits worthy of improvement. Because the RNase P is used as an internal standard, although the steps of sampling and nucleic acid extraction can be effectively monitored, the important link of reverse transcription amplification in the fluorescence RT-PCR cannot be monitored, the reason is that the nucleic acid extracted from the sample contains a large amount of RNase P genes existing in a DNA form, for example, GAPDH or beta-ACTIN genes are used as the internal standard and are similar results, and the main problem is that the design concept of an internal standard primer probe is worthy of improvement. However, as influenza, measles, rubella, new crown and other respiratory infectious diseases are RNA viruses, the first step of fluorescence RT-PCR depends on reverse transcription amplification of reverse transcriptase, which has high temperature sensitivity and is easy to inactivate in the transportation and preservation links of the kit, so that reagent failure is generated to cause result misjudgment and delay epidemic situation control.

Disclosure of the invention

The invention aims to provide a fluorescent RT-PCR (reverse transcription-polymerase chain reaction) specific primer, a probe, a kit and a detection method for detecting a human endogenous RNA (ribonucleic acid) internal standard of a B2M gene, wherein the primer probe of a trans-human Beta 2 Microglobulin (B2M) intron sequence is designed as the internal standard (shown in a schematic diagram in figure 1), the primer and the probe can be effectively amplified only when the B2M gene is transcribed into mRNA (because the length of the intron is about 4000bp base, the amplification product cannot be effectively amplified under a DNA state because of overlong length), the RNA internal standard can monitor the steps of sampling, reverse transcription, nucleic acid extraction, amplification and the like of the fluorescent RT-PCR in the whole process, the quality control of the whole process is realized, and the experimental result can be more reliable.

The technical scheme adopted by the invention is as follows:

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

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

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

B2M fluorescent probe (SEQ ID NO. 3):

ROX-AACGCAAATCCCACTGTCACACTATGCATT-BHQ 2; wherein ROX is a fluorescence reporter group, and BHQ2 is a fluorescence quenching group.

The invention also provides a kit containing the specific primer and the probe, wherein the kit comprises an RT-PCR reaction reagent, the specific primer, the probe, a positive control and a negative control; the positive control is in-vitro transcription RNA of B2M gene, and the negative control is RNase-free H ₂ O。

Preferably, the RT-PCR reaction reagent comprises: 2X 5G qPCR Buffer GB, fluorescent RT-PCR Enzyme Mix, DNase/RNase Free Water.

The invention also provides a method for detecting the B2M gene by using the kit, which comprises the following steps:

(1) Extracting RNA from a sample to be detected; the sample to be detected comprises oropharyngeal swabs, feces, serum and the like;

(2) Adding an RT-PCR reaction reagent, a specific primer and a probe to a sample RNA to be detected as a template for carrying out fluorescence RT-PCR detection; simultaneously, carrying out fluorescence RT-PCR detection of positive control and negative control;

(3) The fluorescence RT-PCR detection takes the Ct value less than 37 and the amplification curve is in the S type as the positive judgment principle; the Ct value is between 37 and 40, and the test is repeated, and the test can obtain a good S-type amplification curve in two times of tests, and the test is judged to be positive.

Preferably, the fluorescent RT-PCR detection conditions are as follows: reverse transcription was performed at 52 ℃ for 5min, pre-denaturation at 95 ℃ for 10s, denaturation at 95 ℃ for 5s, followed by ROX fluorescence detection at 55 ℃ for 20s for 40 cycles.

The key point of the invention is the design of specific primers and probes, and other components in the kit are reaction reagents which are conventionally used for fluorescent RT-PCR in the field, such as dNTP mix and MgCl ₂ The reagent kit comprises a buffer, an enzyme and the like, which can be selected by a person skilled in the art according to common knowledge, can be prepared by the person, and can also be selected from commercial products, such as a Probe 1-step fluorescent RT-PCR 5G kit (TOROIVD, QPR-303) kit and the like of the TOROIVD company, wherein the reaction system is shown in Table 1.

Compared with the prior art, the invention has the following beneficial effects:

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

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

3. The specific primer and the probe can monitor the whole process of sampling, nucleic acid extraction, reverse transcription, PCR and the like, and can provide a novel human endogenous RNA internal standard selection for an infectious disease pathogen fluorescence RT-PCR detection kit.

(IV) description of the drawings

FIG. 1 shows the schematic design of primers and probes specific to the B2M gene.

FIG. 2 shows the result of detecting the fluorescence RT-PCR sensitivity of the specific primers and probes for the B2M gene; a to D are 10 in sequence ⁴ 、10 ³ 、10 ² 10 gene copies.

FIG. 3 shows the results of clinical tests on human pharyngeal swab, blood and stool; and the parts A to C are 5 parts of throat swabs, 5 parts of excrement samples and 3 parts of serum samples in sequence.

(V) detailed description of the preferred embodiments

The invention is further illustrated by the following examples. It is to be understood that these examples are intended only to illustrate the invention and are not intended to limit the scope of the claimed invention, and that no specific experimental conditions or methods are noted in the following examples.

Example 1: design of internal standard primer and probe for specific detection of B2M gene

Searching and downloading the sequence and the gene map of the B2M gene (NG _012920.2, the nucleotide sequence is shown as SEQ ID NO. 4) and the corresponding mRNA (NM _004048.3, the nucleotide sequence is shown as SEQ ID NO. 5) from an NCBI database, and comparing and marking the initial position of the exon by using DNMAN and Snapgene software, wherein the exon 1 is positioned at 1-97bp of the mRNA sequence, the exon 2 is positioned at 3907-4185bp, the exon 3 is positioned at 4813-4840bp, and the exon 4 is positioned at 6091-6629bp.

The method is characterized in that the length of the upstream primer and the downstream primer in a DNA state is about 4000bp, the distance between the upstream primer and the downstream primer in the mRNA state is only about 100bp, and amplification products in the DNA state are too long to effectively amplify, and the design principle 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；

wherein ROX is a fluorescence reporter group, and BHQ2 is a fluorescence quenching group.

Example 2: human endogenous RNA internal standard fluorescence RT-PCR method sensitivity

(1) After the in vitro transcription RNA of the B2M gene is quantified, deionized water is used for 10 times of gradient dilution to obtain a gene sequence of 1 × 10 ⁰ ～1×10 ⁴ copies/. Mu.L for a total of 5 dilutionsIs a standard template;

(2) Taking the RNA obtained by dilution in the step (1) as a template, adding the specific primers and the probes of the embodiment 1 into a PCR tube according to the reaction solution in the table 1, and carrying out reverse transcription real-time fluorescence quantitative PCR detection; the fluorescence amplification curve was obtained, and the results are shown in FIG. 2, in which FIG. 2 is 10 in the order from A to D ⁴ 、10 ³ 、10 ² 10 gene copies, and the lowest detection limit was determined to be 10 copies/reaction.

The positive control is in vitro transcription RNA of a human RNA internal reference B2M gene sequence; negative control was RNase-free H ₂ O。

The amplification reaction conditions are as follows: reverse transcription was performed at 52 ℃ for 5min, pre-denaturation at 95 ℃ for 10s, denaturation at 95 ℃ for 5s, followed by 20s at 55 ℃ for 40 cycles of ROX fluorescence detection at 55 ℃.

Table 1 shows the reagent composition (1 part) of the fluorescent RT-PCR reaction solution

Example 3: human-derived pharyngeal swab, blood, excrement and other clinical sample detection and verification

Randomly selecting 5 pharyngeal swabs, 3 serums and 5 excrement samples stored in a laboratory, wherein the samples are sampled from suspected enterovirus patients and signed with an informed consent during sampling. After the RNA is extracted from the sample, the fluorescence RT-PCR method is adopted for detection by the method of the embodiment 2, good amplification curves can be obtained on the mRNA detection results of B2M, the Ct values of the samples are all less than 37, and the RNA detection results of the B2M gene are all positive (figure 3).

Claims (7)

1. A fluorescent RT-PCR specific primer and probe for detecting a human endogenous RNA internal standard of a B2M gene are characterized in that the specific primer is a B2M upstream primer and a B2M downstream primer, the probe is a B2M fluorescent probe, and the sequences are as follows:

B2M upstream primer: 5 'GGGGGAGGGTTCTCTTCCGC-3';

B2M downstream primer: 5 'GGGCTTTTTTGCGGGAGCGCATG-3';

B2M fluorescent probe: ROX-AACGCAAATCCCACTGTCACACTATGCATT-BHQ 2; wherein ROX is a fluorescence reporter group, and BHQ2 is a fluorescence quenching group.

2. A kit comprising the specific primer and probe of claim 1.

3. The kit of claim 2, wherein the kit comprises RT-PCR reaction reagents, specific primers, probes, positive controls, and negative controls; the positive control is in vitro transcription RNA of B2M gene, and the negative control is RNase-free H ₂ O。

4. The kit of claim 2, wherein the RT-PCR reaction reagents comprise: 2X 5G qPCR Buffer GB, fluorescent RT-PCR Enzyme Mix, DNase/RNase Free Water.

5. A method for detecting B2M gene by using the kit as claimed in claim 1, which comprises the following steps:

(1) Extracting RNA from a sample to be detected;

(2) Adding an RT-PCR reaction reagent, a specific primer and a probe to a sample RNA to be detected as a template for carrying out fluorescence RT-PCR detection; simultaneously, carrying out fluorescence RT-PCR detection of positive control and negative control;

(3) The fluorescence RT-PCR detection takes the Ct value less than 37 and the amplification curve is in the S type as the positive judgment principle; the Ct value is between 37 and 40, and the test is repeated, and the positive result is judged if a good S-type amplification curve can be obtained in two tests.

6. The method of claim 5, wherein the fluorescent RT-PCR detection conditions are: reverse transcription was performed at 52 ℃ for 5min, pre-denaturation at 95 ℃ for 10s, denaturation at 95 ℃ for 5s, followed by ROX fluorescence detection at 55 ℃ for 20s for 40 cycles.

7. The method of claim 5, wherein the test sample comprises oropharyngeal swabs, stool, serum.

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