CN113322351A - Real-time fluorescent quantitative PCR (polymerase chain reaction) probe primer group and kit for rapidly and qualitatively typing and detecting four types of human parainfluenza viruses - Google Patents
Real-time fluorescent quantitative PCR (polymerase chain reaction) probe primer group and kit for rapidly and qualitatively typing and detecting four types of human parainfluenza viruses Download PDFInfo
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Abstract
The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) probe primer group and a kit for quickly, qualitatively and typing and detecting four types of human parainfluenza viruses, wherein the kit comprises a probe primer group specially designed for four types of HPIV-1, HPIV-2, HPIV-3 and HPIV-4 parainfluenza viruses, a primer sequence in the probe primer group is shown as SEQ ID NO.1-27, and a probe sequence in the probe primer group is shown as SEQ ID NO. 28-38. The reaction system of the real-time fluorescent quantitative PCR kit for quickly and qualitatively detecting the four types of human parainfluenza viruses aims at the conserved regions of the four types of human parainfluenza virus genomes, can be used for quickly detecting the parainfluenza viruses in nasopharyngeal swabs or sputum samples, and the detection result can be used for assisting in diagnosing symptoms such as cough, fever, pneumonia and the like caused by the parainfluenza and quickly distinguishing parainfluenza patients from other patients with fever pneumonia.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a real-time fluorescent quantitative PCR probe primer group and a kit for rapidly and qualitatively detecting four types of human parainfluenza viruses by typing.
Background
Human parainfluenza virus (HPIV) is a group of viruses that cause infections of the upper and lower respiratory tract, and is classified into four categories: HPIV-1, HPIV-2, HPIV-3 and HPIV-4 (there are two subtypes, 4a and 4b, of parainfluenza type 4). HPIVs belong to the Paramyxoviridae family (Paramyxoviridae family) in the form of enveloped RNA viruses. HPIVs often cause upper and lower respiratory tract disease in infants, young children, the elderly, and people with weak immune systems, but anyone may become infected. After infection, it takes approximately 2 to 7 days for symptoms to appear. Both HPIV-1 and HPIV-2 cause coughing, with HPIV-1 being one of the most common viruses responsible for colds in infants and young children, and both causing upper and lower respiratory tract illness, as well as cold symptoms. HPIV-3 is more commonly associated with bronchiolitis, bronchitis and pneumonia. Infection by HPIV-4 is less frequent but may result in mild to severe respiratory disease.
Symptoms of upper respiratory disease may include high fever, runny nose, cough, and symptoms of lower respiratory disease may include throat (vocal cords (larynx), tracheal and bronchial infections), bronchitis (major airway infection connecting the trachea and the lungs), bronchiolitis (lung minimal airway infection), pneumonia (lung infection). Other symptoms of HPIV disease may include: sore throat, sneezing, wheezing, ear pain, irritability, and decreased appetite.
HPIVs can be transmitted from an infected person to a healthy person by coughing or sneezing, among other means. Close personal contact, such as touching or shaking hands, or touching an object or surface with HPIV, and then touching the mucous membrane such as the mouth, nose or eyes, can also be infected.
The four types of HPIV have different epidemiological characteristics and cycle at different times of the year. Studies according to the american epidemiology show that: HPIV-1, which causes cough in children, is often more frequently seen in autumn of odd years. HPIV-2, which can cause pharyngolaryngitis, is more common in the fall each year. The infection frequency is lower than that of HPIV-1 and HPIV-3. HPIV-3 infection usually occurs in the spring and early summer months of the year. However, HPIV-3 infection may occur throughout the year, particularly when HPIV-1 and HPIV-2 are not seasonal. The seasonal pattern of HPIV-4 (subtypes 4a and 4b) is not well described at present.
Currently, there is no accurate number of patients with parainfluenza, but many researchers consider that the number of parainfluenza patients per year is quite high. Parainfluenza is common in the fall and winter seasons of the year, with symptoms being most severe in infants and progressively milder with age. Most of the school-age children have a history of parainfluenza virus exposure, and most people in the adult population already have antibodies to parainfluenza. However, repeated infection with parainfluenza virus may lead to serious lower respiratory tract diseases such as pneumonia, bronchitis, and bronchiolitis. Elderly and persons with compromised immune systems are at higher risk of serious infections.
Parainfluenza virus diagnosis methods include: direct detection of viral genomes using Polymerase Chain Reaction (PCR); directly detecting viral antigens in respiratory secretions (collected within 1 week after symptoms appear) using immunofluorescence or enzyme immunoassay; separating and identifying virus in cell culture; HPIV-specific IgG antibody levels were significantly elevated in serum samples. No real-time fluorescent quantitative PCR kit for detecting four types of human parainfluenza viruses by rapid qualitative typing is available in the prior art.
Disclosure of Invention
Aiming at the technical problems in the related art, the invention provides a real-time fluorescent quantitative PCR probe primer group and a kit for rapidly and qualitatively detecting four types of human parainfluenza viruses, which can overcome the defects in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a real-time fluorescent quantitative PCR probe primer group for rapidly and qualitatively detecting four types of human parainfluenza viruses comprises a probe primer group specially designed for the four types of the parainfluenza viruses including HPIV-1, HPIV-2, HPIV-3 and HPIV-4, wherein the primer sequence in the probe primer group is as follows:
HPIV-1-F1 is shown as SEQ ID NO. 1;
HPIV-1-R1 is shown as SEQ ID NO. 2;
HPIV-2-F1 is shown as SEQ ID NO. 3;
HPIV-2-R1 is shown as SEQ ID NO. 4;
HPIV-3-F1 is shown as SEQ ID NO. 5;
HPIV-3-R1 is shown as SEQ ID NO. 6;
HPIV-4-F1 is shown as SEQ ID NO. 7;
HPIV-4-R1 is shown as SEQ ID NO. 8;
HPIV-4-F2 is shown as SEQ ID NO. 9;
HPIV-4-R2 is shown as SEQ ID NO. 10;
HPIV-4-R3 is shown as SEQ ID NO. 11;
HPIV-4-F3 is shown as SEQ ID NO. 12;
HPIV-4-F4 is shown as SEQ ID NO. 13;
HPIV-4-F5 is shown as SEQ ID NO. 14;
HPIV-4-R4 is shown as SEQ ID NO. 15;
HPIV-4-F6 is shown as SEQ ID NO. 16;
HPIV-4-R5 is shown as SEQ ID NO. 17;
HPIV-4-F7 is shown as SEQ ID NO. 18;
HPIV-3-R2 is shown as SEQ ID NO. 19;
HPIV3-P3-F1 is shown in SEQ ID NO. 20;
HPIV3-P3-R1 is shown in SEQ ID NO. 21;
HPIV4-P3-F1 is shown in SEQ ID NO. 22;
HPIV4-P3-R1 is shown in SEQ ID NO. 23;
HPIV3-P3-F2 is shown in SEQ ID NO. 24;
HPIV3-P3-F3 is shown in SEQ ID NO. 25;
HPIV3-P3-F4 is shown in SEQ ID NO. 26;
HPIV3-P3-R2 is shown in SEQ ID NO. 27;
the probe sequence in the probe primer group is as follows:
HPIV-1-P1 is shown as SEQ ID NO. 28;
HPIV-2-P1 is shown as SEQ ID NO. 29;
HPIV-3-P1 is shown as SEQ ID NO. 30;
HPIV-4-P1 is shown as SEQ ID NO. 31;
HPIV-1-P2 is shown as SEQ ID NO. 32;
HPIV-3-P2 is shown as SEQ ID NO. 33;
HPIV-1-P2 is shown as SEQ ID NO. 34;
HPIV-2-P2 is shown as SEQ ID NO. 35;
HPIV-3-P2 is shown as SEQ ID NO. 36;
HPIV3-P3 is shown as SEQ ID NO. 37;
HPIV4-P3 is shown in SEQ ID NO. 38.
Further, the 5' end of the probe in the probe primer group is labeled with FAM, VIC or NED.
Further, the 3' ends of the probes in the probe primer group are all marked with MGB.
According to another aspect of the invention, a real-time fluorescent quantitative PCR kit for rapid qualitative typing detection of four types of human parainfluenza viruses is provided, which comprises a probe primer group specially designed for HPIV-1, HPIV-2, HPIV-3 and HPIV-4, wherein the primer sequence in the probe primer group is shown as SEQ ID NO.1-27, and the probe sequence in the probe primer group is shown as SEQ ID NO. 28-38.
Further, the real-time fluorescence quantitative PCR kit for rapid qualitative typing detection of the four types of human parainfluenza viruses further comprises reverse transcriptase, high-efficiency DNA polymerase, dNTPs, qPCR buffer solution suitable for quantitative analysis, ROX reference fluorescent dye, blank control and positive control.
Further, when the corresponding FAM, VIC or NED channel is detected to have an obvious S-type amplification curve, the sample to be detected contains HPIV-1, HPIV-2, HPIV-3 or HPIV-4a/4 b.
Further, after setting a threshold value according to the signal maximum value of the blank control, detecting that the corresponding FAM, VIC or NED channel has no S-type amplification curve or has a weaker amplification curve and the Ct value is greater than 38, and determining that the sample to be detected is negative.
The invention has the beneficial effects that: the reaction system of the real-time fluorescent quantitative PCR kit for rapidly and qualitatively detecting the four types of human parainfluenza viruses aims at the conserved regions of the four types of human parainfluenza virus genomes, can be used for rapidly detecting the parainfluenza viruses in nasopharyngeal swabs or sputum samples, and the detection result can be used for assisting in diagnosing symptoms such as cough, fever, pneumonia and the like caused by parainfluenza and rapidly distinguishing parainfluenza patients from other patients with fever pneumonia; the reaction system can be used for extracting a good nucleic acid sample and can also be directly used for preserving fluid containing virus particles, the nucleic acid is not required to be extracted for direct detection, the time and the reagent cost are saved, the experimental result is easy and convenient to interpret, and the operator is not required to be trained additionally.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing the results of an HPIV-1 assay according to an embodiment of the present invention;
FIG. 2 is a second graph of the results of an HPIV-1 assay according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of an HPIV-2 assay according to an embodiment of the present invention;
FIG. 4 is a second graph of the results of an HPIV-2 assay according to an embodiment of the present invention;
FIG. 5 is a graph showing the results of an HPIV-3 assay according to an embodiment of the present invention;
FIG. 6 is a second graph of the results of an HPIV-3 type assay according to an embodiment of the present invention;
FIG. 7 is a graph showing the results of an HPIV-4 assay according to an embodiment of the present invention;
FIG. 8 is a second graph of the results of an HPIV-4 assay according to an embodiment of the present invention;
FIG. 9 is a graph showing the results of a mixed extraction-free assay according to embodiments of the present invention for HPIV-1, 2 and 3 types;
FIG. 10 is a second graph of the results of HPIV-1, 2 and 3 mixed non-extraction assay according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1
A real-time fluorescent quantitative PCR kit for rapidly, qualitatively and typing and detecting four types of human parainfluenza viruses (including HPIV-1, HPIV-2, HPIV-3 and HPIV-4a/4b) comprises reverse transcriptase, high-efficiency DNA polymerase, dNTPs, a probe primer group specially designed for the four types of parainfluenza viruses, qPCR buffer solution suitable for quantitative analysis, ROX reference fluorescent dye, blank control and positive control. The primer sequences in the probe primer group are shown in table 1, and the probe sequences in the probe primer group are shown in table 2.
TABLE 1 primer sequences
TABLE 2 Probe sequences
The detection method by adopting the kit comprises the following steps:
(1) as shown in Table 3, a fluorescent quantitative PCR reaction system was prepared.
TABLE 3 fluorescent quantitative PCR reaction System
(2) The fluorescent quantitative PCR reaction program (adapted to ABI 7500 and StepOne series) is shown in Table 4.
TABLE 4 fluorescent quantitative PCR reaction procedure
(3) The experimental results are as follows:
(a) preparing HPIV quality control products:
to verify the effectiveness of the present invention, human parainfluenza virus ribonucleic acid type 1-4 (HPIV RNA) liquid indoor quality control products manufactured by Bangdish bioscience, Guangzhou were purchased. The copy number was about 1.55E + 05/mL.
(b) HPIV-1 type detection experiment:
4 portions of HPIV-1 type quality control product were taken, and nucleic acid was extracted using a "full-automatic nucleic acid extractor" of Jiangsu Shuichio Biotech Co., Ltd. Adding the extracted nucleic acid (2uL) into a prepared fluorescent quantitative PCR reaction system, and amplifying according to a standard reaction program. The experimental results are shown in FIGS. 1-2.
Wherein the negative control has no signal, and the 4 reaction wells to be detected have obvious signal amplification. The Ct values are shown in Table 5:
TABLE 5 HPIV-1 type assay results
(c) HPIV-2 type detection experiment:
4 portions of HPIV-2 type quality control product were taken, and nucleic acid was extracted using a "full-automatic nucleic acid extractor" of Jiangsu Shuichio Biotech Co., Ltd. Adding the extracted nucleic acid (2uL) into a prepared fluorescent quantitative PCR reaction system, and amplifying according to a standard reaction program. The experimental results are shown in FIGS. 3-4.
Wherein the negative control has no signal, and the 4 reaction wells to be detected have obvious signal amplification. The Ct values are shown in Table 6:
TABLE 6 HPIV-2 type assay results
| Well | Sample Name | Reporter | Quencher | Ct value |
| A4 | HPIV-2 quality control product | VIC | NFQ-MGB | 30.23 |
| B4 | HPIV-2 quality control product | VIC | NFQ-MGB | 30.17 |
| C4 | HPIV-2 quality control product | VIC | NFQ-MGB | 32.60 |
| D4 | HPIV-2 quality control product | VIC | NFQ-MGB | 29.31 |
| F4 | Purified water | VIC | NFQ-MGB | Undetermined |
| G4 | Human DNA | VIC | NFQ-MGB | Undetermined |
| H4 | Tap water | VIC | NFQ-MGB | Undetermined |
(d) HPIV-3 type detection experiment:
3 portions of HPIV-3 type quality control product were taken, and nucleic acid was extracted using a "full-automatic nucleic acid extractor" of Jiangsu Shuichio Biotech Co., Ltd. Adding the extracted nucleic acid (2uL) into a prepared fluorescent quantitative PCR reaction system, and amplifying according to a standard reaction program. The results are shown in FIGS. 5-6.
Wherein the negative control has no signal, and the 3 reaction wells to be detected have obvious signal amplification. The Ct values are shown in Table 7:
TABLE 7 HPIV-3 type test results
| Well | Sample Name | Reporter | Quencher | Ct value |
| A2 | HPIV-3 quality control product | NED | NFQ-MGB | 16.61 |
| B2 | HPIV-3 quality control product | NED | NFQ-MGB | 16.62 |
| C2 | HPIV-3 quality control product | NED | NFQ-MGB | 16.34 |
| F2 | Purified water | NED | NFQ-MGB | Undetermined |
| G2 | Human DNA | NED | NFQ-MGB | Undetermined |
| H2 | Tap water | NED | NFQ-MGB | Undetermined |
(e) HPIV-4 type detection experiment:
3 parts of each of the HPIV-4a and HPIV-4b quality control products were taken, and nucleic acid was extracted using a "full-automatic nucleic acid extractor" of Jiangsu Shuichio Biotech Co., Ltd. Adding the extracted nucleic acid (2uL) into a prepared fluorescent quantitative PCR reaction system, and amplifying according to a standard reaction program. The experimental results are shown in FIGS. 7-8.
Wherein the negative control has no signal, and the 6 reaction wells to be detected have obvious signal amplification. The Ct values are shown in Table 8:
TABLE 8 HPIV-4 type assay results
| Well | Sample Name | Target Name | Reporter | Quencher | Ct value |
| B1 | HPIV-4a quality control product | HPIV-4 | FAM | NFQ-MGB | 22.70 |
| C1 | HPIV-4a quality control product | HPIV-4 | FAM | NFQ-MGB | 22.61 |
| D1 | HPIV-4a quality control product | HPIV-4 | FAM | NFQ-MGB | 23.15 |
| E1 | HPIV-4b quality control product | HPIV-4 | FAM | NFQ-MGB | 22.92 |
| F1 | HPIV-4b quality control product | HPIV-4 | FAM | NFQ-MGB | 23.33 |
| H1 | HPIV-4b quality control product | HPIV-4 | FAM | NFQ-MGB | 22.88 |
| B2 | Purified water | HPIV-4 | FAM | NFQ-MGB | Undetermined |
| B3 | Purified water | HPIV-4 | FAM | NFQ-MGB | Undetermined |
| A2 | Human DNA | HPIV-4 | FAM | NFQ-MGB | Undetermined |
| A3 | Human DNA | HPIV-4 | FAM | NFQ-MGB | Undetermined |
(f) HPIV-1, 2 and 3 mixed extraction-free detection experiments:
the HPIV-1, 2 and 3 types of quality control materials were each 1 part and diluted ten times with purified water. Respectively adding 2uL of the diluted quality control material into a prepared fluorescent quantitative PCR reaction system, and carrying out amplification according to a standard reaction program (2 reaction holes). The experimental results are shown in FIGS. 9-10.
The signal of three probes in each of the two wells to be tested has Ct values as shown in Table 9:
TABLE 9 HPIV-1, 2 and 3 Mixed non-extraction test results
In conclusion, by means of the technical scheme, the reaction system of the real-time fluorescence quantitative PCR kit for rapidly and qualitatively detecting four types of human parainfluenza viruses aims at the conserved regions of four types of human parainfluenza virus genomes, can be used for rapidly detecting the parainfluenza viruses in nasopharyngeal swabs or sputum samples, and the detection result can be used for assisting in diagnosing symptoms such as cough, fever and pneumonia caused by parainfluenza and rapidly distinguishing parainfluenza patients from other patients with fever and pneumonia; the reaction system can be used for extracting a good nucleic acid sample and can also be directly used for preserving fluid containing virus particles, the nucleic acid is not required to be extracted for direct detection, the time and the reagent cost are saved, the experimental result is easy and convenient to interpret, and the operator is not required to be trained additionally. When the kit is used for detection, when nucleic acid (RNA) of four parainfluenza viruses (including HPIV-1, HPIV-2, HPIV-3 and HPIV-4a/4b) is used as a template, a corresponding probe channel (FAM, VIC or NED) can be detected to have an obvious S-shaped amplification curve, and after a threshold value is set according to the signal maximum value of a blank control, a negative sample has no S-shaped amplification curve or has a weaker amplification curve and the Ct value is larger than 38.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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Claims (7)
1. A real-time fluorescent quantitative PCR probe primer group for rapidly and qualitatively detecting four types of human parainfluenza viruses is characterized by comprising probe primer groups aiming at HPIV-1, HPIV-2, HPIV-3 and HPIV-4, wherein the primer sequences in the probe primer groups are shown as SEQ ID NO.1-27, and the probe sequences in the probe primer groups are shown as SEQ ID NO. 28-38.
2. The real-time fluorescent quantitative PCR probe primer set for the rapid qualitative typing detection of four types of human parainfluenza viruses according to claim 1, wherein the 5' end of the probe in the probe primer set is labeled with FAM, VIC or NED.
3. The real-time fluorescent quantitative PCR probe primer set for the rapid qualitative typing detection of four types of human parainfluenza viruses as claimed in claim 1, wherein the 3' ends of the probes in the probe primer set are all labeled with MGB.
4. A real-time fluorescent quantitative PCR kit for rapidly and qualitatively detecting four types of human parainfluenza viruses is characterized by comprising probe primer groups aiming at HPIV-1, HPIV-2, HPIV-3 and HPIV-4, wherein the primer sequences in the probe primer groups are shown as SEQ ID NO.1-27, and the probe sequences in the probe primer groups are shown as SEQ ID NO. 28-38.
5. The real-time fluorescent quantitative PCR kit for rapid qualitative typing detection of four types of human parainfluenza viruses according to claim 4, characterized in that it further comprises reverse transcriptase, DNA polymerase, dNTPs, qPCR buffer, ROX reference fluorescent dye, blank control and positive control.
6. The real-time fluorescent quantitative PCR kit for rapid qualitative typing detection of four types of human parainfluenza viruses according to claim 4, wherein when a significant S-type amplification curve is detected in a corresponding FAM, VIC or NED channel, the sample to be detected contains HPIV-1, HPIV-2, HPIV-3 or HPIV-4a/4 b.
7. The real-time fluorescent quantitative PCR kit for rapid qualitative typing detection of four types of human parainfluenza viruses according to claim 4, wherein after a threshold is set according to the signal maximum value of the blank control, the corresponding FAM, VIC or NED channel is detected without S-type amplification curve, or when the corresponding FAM, VIC or NED channel has a weaker amplification curve and the Ct value is greater than 38, the sample to be detected is negative.
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| CN113462656A (en) * | 2021-03-24 | 2021-10-01 | 兰州生物制品研究所有限责任公司 | Human three-type parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof |
| CN113817870A (en) * | 2021-09-10 | 2021-12-21 | 宁波海尔施基因科技有限公司 | Primer composition for simultaneously detecting seven respiratory tract-related viruses and application thereof |
| CN114480741A (en) * | 2022-02-23 | 2022-05-13 | 深圳市赛格诺生物科技有限公司 | Oligonucleotide mixture, fluorescent RT-PCR detection reagent and application |
| CN116479175A (en) * | 2023-03-13 | 2023-07-25 | 广州国家实验室 | Detection system, kit and method for detecting human parainfluenza virus nucleic acid |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113462656A (en) * | 2021-03-24 | 2021-10-01 | 兰州生物制品研究所有限责任公司 | Human three-type parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof |
| CN113462656B (en) * | 2021-03-24 | 2022-09-30 | 兰州生物制品研究所有限责任公司 | Human three-type parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof |
| CN113817870A (en) * | 2021-09-10 | 2021-12-21 | 宁波海尔施基因科技有限公司 | Primer composition for simultaneously detecting seven respiratory tract-related viruses and application thereof |
| CN113817870B (en) * | 2021-09-10 | 2023-12-22 | 宁波海尔施基因科技股份有限公司 | Primer composition for simultaneously detecting seven respiratory tract related viruses and application thereof |
| CN114480741A (en) * | 2022-02-23 | 2022-05-13 | 深圳市赛格诺生物科技有限公司 | Oligonucleotide mixture, fluorescent RT-PCR detection reagent and application |
| CN116479175A (en) * | 2023-03-13 | 2023-07-25 | 广州国家实验室 | Detection system, kit and method for detecting human parainfluenza virus nucleic acid |
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