CN114836580A

CN114836580A - Multiplex qPCR detection primer combination for respiratory infectious disease pathogens

Info

Publication number: CN114836580A
Application number: CN202210617046.2A
Authority: CN
Inventors: 冯悦; 张伊菲; 夏雪山; 刘丽
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2022-08-02
Anticipated expiration: 2042-06-02

Abstract

The invention discloses a multiple qPCR detection primer combination for pathogens of respiratory infectious diseases, which comprises specific primers and probes for detecting influenza A virus, influenza B virus, parainfluenza virus 1, parainfluenza virus 3, respiratory syncytial virus A, rhinovirus, metapneumovirus and respiratory syncytial virus B; the invention uses real-time fluorescence quantitative PCR technology to detect pathogen target gene; the method has the advantages of short time consumption, high specificity and low cost, can detect various pathogens at one time, provides a convenient method for detecting the pathogens of the respiratory infectious diseases, and has important significance for the early clinical molecular diagnosis of respiratory infectious patients.

Description

Multiplex qPCR detection primer combination for respiratory infectious disease pathogens

Technical Field

The invention belongs to the technical field of pathogen detection, and relates to a multiple qPCR detection primer combination for respiratory infectious disease pathogens.

Background

Respiratory tract infection is a common clinical disease and also a main lethal disease, and can be caused by various microorganisms such as viruses, bacteria, fungi, mycoplasma, chlamydia and the like, so that the respiratory tract infection has strong infectivity and various clinical symptoms.

Acute Upper Respiratory Infection (URTI) can cause common cold, acute viral pharyngitis, laryngitis, acute tonsillitis and other common clinical symptoms. Children, the elderly, people with low immune function and/or respiratory diseases are more susceptible to infection. The patients with the diseases often have the clinical characteristics of short course of disease, easy self-healing, good prognosis and the like. A few patients with acute upper respiratory tract infection are accompanied with serious complications, such as upper respiratory tract infection represented by pharyngitis, and some patients can be secondarily suffered from rheumatic fever and glomerulonephritis caused by hemolytic streptococcus; a small number of patients may also develop viral myocarditis, acute sinusitis, otitis media, tracheo-bronchitis, etc.

Acute Lower Respiratory Tract Infections (LRTI) are common in cold seasons, often causing clinical symptoms including acute bronchitis, acute bronchiolitis, and pneumonia. Acute lower respiratory infection is an important factor in the death of children under 5 years of age in developing countries, and according to data published by WHO 2018, lower respiratory infection in 2016 causes 300 million deaths worldwide. Early, timely and accurate diagnosis is helpful for accurate clinical medication, helps patients to recover health, and reduces the fatality rate. In view of this, it is necessary to enhance the diagnosis of pathogens of clinically common respiratory infectious diseases such as influenza virus, parainfluenza virus, rhinovirus, and the like.

Respiratory pathogen detection methods are numerous. The traditional bacteria or fungi culture identification method is time-consuming and low in detection rate; the commercial test strip (kit) based on the antigen and antibody reaction principle has the advantages of simplicity, convenience and rapidness, but the application is limited due to low sensitivity and false positive results. In recent years, the molecular biology related detection technology is vigorously developed, and the technologies such as PCR-first generation sequencing, LAMP, gene chip, digital PCR, NGS and the like are all used for detecting respiratory pathogens; however, the above-mentioned technical method still has the disadvantages of high price, high operation requirement, time consumption and the like.

Disclosure of Invention

The invention provides a multiple qPCR detection primer combination for respiratory infectious disease pathogens, which comprises specific primers and probes for detecting 8 respiratory infectious disease pathogens, and also relates to other conventional detection reagents for multiple qPCR detection in the multiple qPCR detection.

The multiplex qPCR detection primer combination comprises specific primers and probes for detecting influenza A virus, influenza B virus, parainfluenza virus 1, parainfluenza virus 3, respiratory syncytial virus A, rhinovirus, metapneumovirus and respiratory syncytial virus B; wherein the specific primers are SEQ ID NO 1 and SEQ ID NO 2 for influenza A virus, SEQ ID NO 4 and SEQ ID NO 5 for influenza B virus, SEQ ID NO 7 and SEQ ID NO 8 for parainfluenza virus type 1, SEQ ID NO 10 and SEQ ID NO 11 for parainfluenza virus type 3, SEQ ID NO 13 and SEQ ID NO 14 for respiratory syncytial virus type A, SEQ ID NO 16 and SEQ ID NO 17 for rhinovirus, SEQ ID NO 19 and SEQ ID NO 20 for metapneumovirus, and SEQ ID NO 22 and SEQ ID NO 23 for respiratory syncytial virus type B; the probes are SEQ ID NO 3 for influenza A virus, SEQ ID NO 6 for influenza B virus, SEQ ID NO 9 for parainfluenza virus type 1, SEQ ID NO 12 for parainfluenza virus type 3, SEQ ID NO 15 for respiratory syncytial virus type A, SEQ ID NO 18 for rhinovirus, SEQ ID NO 21 for metapneumovirus, and SEQ ID NO 24 for respiratory syncytial virus type B.

The method for detecting primer combinations using the multiplex qPCR described above was as follows:

1. extracting nucleic acid (RNA) of a sample, wherein the sample is a throat swab or a nose swab;

A. putting the collected nasal swab or throat swab sample into a preservation tube containing 1mL of virus preservation solution, and fully oscillating to promote pathogen release;

B. putting 200 mu L of pathogen preservation solution in the step A into a 1.5mL centrifuge tube, and then adding 500 mu L of cell lysate for lysis;

C. taking 700 mu L of lysate in the step B, and extracting nucleic acid by using a Tiangen virus DNA/RNA genome extraction kit;

2. detecting by using the nucleic acid in the step (1) as a template and adopting specific primers and probes targeting 8 pathogens through multiple real-time fluorescent quantitative PCR, taking a human respiratory epithelial cell ribonuclease P gene as an internal reference, and performing result judgment according to a Ct value;

specific primers and probes of influenza A virus, influenza B virus, parainfluenza virus type 1 and parainfluenza virus type 3 are commonly used in detection; specific primers and probes of respiratory syncytial virus A, rhinovirus, metapneumovirus and respiratory syncytial virus B are used together in detection, and a human respiratory epithelial cell ribonuclease P gene is used as an internal reference;

the nucleotide sequences of the specific primers and probes for detecting 8 pathogens and the reference genes are shown as SEQ ID NO. 1-SEQ ID NO. 27;

the reaction system and the reaction conditions for the multiplex real-time fluorescent quantitative PCR detection are as follows:

amplification conditions: at 55 ℃ for 30 min; fluorescence was collected at 95 ℃, 30s, 95 ℃, 5s, 58 ℃, 30s (45 cycles);

3. the detection result interpretation comprises the following steps:

(1) ct value of internal reference (human respiratory tract epithelial cell ribonuclease P gene, RNP gene) is less than or equal to 36, and Ct value of negative control group and template-free control group is not greater; if the detection result is not qualified, performing multiplex real-time fluorescence quantitative PCR detection again, or re-extracting nucleic acid to perform multiplex real-time fluorescence quantitative PCR detection;

(2) the Ct value of the pathogen is less than or equal to 36.0, and if the Ct value is greater than 36.0, single real-time fluorescent quantitative PCR verification needs to be carried out on the pathogen;

(3) the amplification curve is in a standard S shape and has no abnormal fluctuation.

Compared with the prior art, the invention has the following advantages and technical effects:

1. the primer and the probe set for detecting 8 respiratory infectious disease pathogens provided by the invention have the advantages of high detection efficiency and accurate detection result, and can quickly finish pathogen diagnosis at low cost;

2. the invention carries out qualitative detection on the specific target gene of the pathogen on the basis of a real-time fluorescent quantitative PCR technical platform, can detect a plurality of samples in one experiment, has the characteristics of rapidness, specificity, economy and the like, and greatly reduces the detection cost of respiratory tract infectious samples.

Drawings

FIG. 1 is a graph of single fluorescent quantitative PCR amplification of influenza A virus;

FIG. 2 is a graph of single fluorescent quantitative PCR amplification curve of influenza B virus;

FIG. 3 is a graph of a single fluorescent quantitative PCR amplification plot of parainfluenza virus type 1;

FIG. 4 is a graph of a single fluorescent quantitative PCR amplification plot of parainfluenza virus type 3;

FIG. 5 is a graph of single fluorescent quantitative PCR amplification curve for respiratory syncytial virus type A;

FIG. 6 is a graph of single fluorescent quantitative PCR amplification of rhinovirus;

FIG. 7 is a graph of single fluorescent quantitative PCR amplification of metapneumovirus;

FIG. 8 is a graph of a single fluorescent quantitative PCR amplification curve for respiratory syncytial virus type B;

FIG. 9 is a graph of a single fluorescent quantitative PCR amplification curve of an internal reference RNP gene;

FIG. 10 shows the results of multiplex qPCR specific assays for influenza A, B, parainfluenza virus type 1, parainfluenza virus type 3;

FIG. 11 shows the results of multiplex qPCR specific assays for respiratory syncytial virus type A, rhinovirus, metapneumovirus, respiratory syncytial virus type B;

FIG. 12 is the results of multiple qPCR sensitivity tests for influenza A and B viruses;

FIG. 13 is the results of a multiplex qPCR sensitivity assay for parainfluenza virus type 1 and parainfluenza virus type 3;

FIG. 14 is the results of a multiplex qPCR sensitivity assay for respiratory syncytial virus type A and rhinovirus;

FIG. 15 is the results of multiplex qPCR sensitivity assays for metapneumovirus and respiratory syncytial virus type B;

FIG. 16 shows the results of multiplex qPCR sensitivity test for reference RNP gene.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by means of specific embodiments, but the present invention is not limited to the scope of the embodiments; the materials used in the following examples are not limited to those listed above, and other similar materials may be substituted, and the reagents and methods used in the examples, according to the conventional conditions or conditions recommended by the manufacturers, without specifying the apparatus, may be the conventional reagents and methods.

Example 1: design of specific primers and probes

1. The pathogen gene reference sequences were downloaded in the NCBI (National Center for Biotechnology Information, National Center for Biotechnology) website as follows:

20 genes each of which encodes influenza A virus matrix protein 1 (M1) and matrix protein 2 (M2), influenza B virus matrix protein 1 (M1) and BM2 protein (BM2), parainfluenza virus type 1-hemagglutinin-neuroaminidase (HN) and parainfluenza virus type 3-hemagglutinin-neuroaminidase (HN) respectively, respiratory syncytial virus A matrix protein (M) and rhinovirus polyprotein gene respectively, metapneumovirus nucleotein (N) and syncytial B virus matrix protein (M); the alignment of nucleotide sequences was performed using Mega 5.0 software, primers and probes were designed using Primer Select software, and the following conditions were required:

(1) tm value: the Tm value of the probe is 8-10 ℃ higher than that of the primer, wherein the Tm value of the probe is 60 ℃ in general;

(2) GC content: generally not less than 40%;

(3) primer dimer was not generated and the hairpin structure software evaluation result was OK;

(4) the size of the amplified fragment is generally less than 200 bp;

2. the preliminarily designed primer probe nucleotide sequences are compared by using the BLAST retrieval function in the NCBI website again, and primers and probe sequences with high specificity are selected;

the nucleotide sequences of the specific primers and probes targeting 8 respiratory infectious disease pathogens and internal reference RNP genes are shown in SEQ ID NO 1-SEQ ID NO 27 and shown in the following table;

。

example 2: establishment of real-time fluorescent quantitative PCR method

1. Plasmid construction

Connecting specific sequences of 8 pathogens (shown in SEQ ID NO:28 and SEQ ID NO: 36) and sequences of an internal reference gene GAPDH with a pUC57 vector to synthesize a plasmid standard product, wherein influenza A virus synthesizes a plasmid, influenza B virus and parainfluenza virus 1 synthesize a plasmid, parainfluenza virus 3 and RNP synthesize a plasmid, respiratory syncytial virus A and rhinovirus synthesize a plasmid, and metapneumovirus and respiratory syncytial virus B synthesize a plasmid; the plasmid construction is completed by Zhongmeitai and the biotechnology Beijing GmbH; measuring the concentration by an ultraviolet spectrophotometer, and calculating the copy number of the plasmids according to the length and the concentration of each plasmid;

copy number results are shown in the table below;

the plasmid was diluted in 10-fold dilution gradient, andsetting 8 gradients to 10 respectively ⁷ 、10 ⁶ 、10 ⁵ 、10 ⁴ 、10 ³ 、10 ² 、10 ¹ 、10 ⁰ On the order of copies/. mu.L.

2. Specificity, sensitivity and repeatability experiments

(1) Specificity of

The single real-time fluorescent quantitative PCR has the following reaction system and conditions:

the plasmid is used as a template to carry out a single real-time fluorescent quantitative PCR experiment, and the configuration of a reaction system and the setting conditions of the reaction are as follows:

amplification conditions: 30sec at 95 ℃; (95 ℃ 5sec, 58 ℃ 30sec fluorescence collection) (45 cycles);

taking the synthesized plasmid standard substance as a positive substance, performing single-fold fluorescence quantitative PCR experiment (reaction system and amplification conditions are as follows) by using a one-step method RT-qPCR kit of Novozan Bio, and simultaneously detecting plasmid mixed samples (with gradient dilution concentration of 10) of the 8 respiratory pathogens and 1 internal reference RNP genes ⁵ copies/μL-10 ³ copies/. mu.L); the results of the single fluorescent quantitative PCR experiments of the pathogens are shown in the figures 1-9, and the amplification results show that the 8 pathogens and the internal reference RNP gene have amplification curves and have no nonspecific amplification, which indicates that the designed primers and probes have good specificity.

The multiplex real-time fluorescence quantitative PCR comprises the following reaction systems and conditions:

amplification conditions: 30sec at 95 ℃; (fluorescence collected at 95 ℃ for 5sec, 58 ℃ for 30 sec.) 45 cycles;

plasmid mixed samples (gradient dilution concentration of 10) for simultaneously detecting the 8 respiratory pathogens and the 1 internal reference RNP genes by taking the synthesized plasmid standard substance as a positive substance ⁵ copies/μL-10 ³ copies/μL) (ii) a The results of the pathogen multiple fluorescence quantitative PCR experiments (reaction system and amplification conditions are as follows) are shown in FIGS. 10-11, and the amplification results show that the 8 pathogens and the internal reference RNP gene have amplification curves, and the primers and the probes cannot influence each other after being combined.

(2) Sensitivity of the probe

Multiplex qPCR method on serial concentration gradient diluted 10 ⁷ 、10 ⁶ 、10 ⁵ 、10 ⁴ 、10 ³ 、10 ² 、10 ¹ 、10 ⁰ Detecting a plasmid template with the order of copies/mu L, and determining the lowest plasmid copy value which can be detected by the multiple qPCR detection method; the results are shown in FIGS. 12-16, wherein the detection limit of influenza A virus, influenza B virus, parainfluenza virus 1, parainfluenza virus 3, respiratory syncytial virus A, respiratory syncytial virus B and internal reference RNP reaches 10 copies/mu L; the detection limit of rhinovirus and metapneumovirus is 10 ² On the order of copies/. mu.L.

(3) Repeatability of

To verify the stability of the established multiplex qPCR detection method, 10 was used ³ Experiments are carried out on plasmids with the order of copies/mu L, and batch-to-batch repeatability experiments are respectively carried out; the plasmid was subjected to a fluorescent quantitative PCR experiment using specific primers and probe sets for each group of pathogens, and CV values were calculated for the batch and three consecutive weeks of the batch experiment, with the following repeatability results:

according to the result of repeated experiments, the established multiple real-time fluorescence quantitative method based on 8 respiratory infectious disease pathogens and 1 internal reference gene has higher stability (CV is less than 5%).

Example 3: sample nucleic acid (RNA) extraction

In this example, RNA was extracted from throat or nasal swabs of respiratory tract infected patients using a commercial TIANAmp Virus DNA/RNA Kit (TIANGEN, Beijing) as follows:

A. preparation of Carrier RNA aqueous solution: suck 310 muL enzyme-free ddH ₂ Dissolving 310 mu g of Carrier RNA dry powder in O, fully shaking, and then centrifuging briefly to obtain a Carrier RNA aqueous solution with the final concentration of 1 ng/mu L, and storing at low temperature for later use;

B. preparing Carrier RNA working solution: adding buffer solution GB, Carrier RNA aqueous solution and proteinase K into the samples according to the number (n) of the extracted samples, wherein the volumes of the buffer solution GB, the Carrier RNA aqueous solution and the proteinase K are respectively 0.22mL multiplied by n, 6.16 mu L multiplied by n and 20 mu L multiplied by n, oscillating, fully mixing uniformly, and then centrifuging briefly to collect wall-hanging liquid;

C. extracting a sample and preprocessing: the indoor quality control product is liquid and does not need to be treated, and the inactivated strain and the strain are diluted by adding 1mL of normal saline; sucking 200 mu L of the uniformly mixed sample and 220 mu L of Carrier RNA working solution, adding the uniformly mixed sample and the Carrier RNA working solution into a 1.5mL enzyme-free centrifuge tube, oscillating, fully mixing, and then centrifuging briefly to collect wall-hanging liquid;

D. and (3) incubation: placing the mixed liquid in a 56 ℃ water bath, incubating for 15 minutes, and manually shaking and uniformly mixing once every 5 minutes; wiping paper to suck water on the outer wall of the centrifuge tube, and centrifuging briefly to collect wall-hung liquid; 0.25mL of absolute ethyl alcohol is absorbed and added into a centrifuge tube, after oscillation for 15 seconds and full mixing, the mixture is kept stand for 5 minutes at room temperature, and the wall-hanging liquid is collected by short centrifugation;

E. transferring: transferring all liquid in the centrifuge tube into a CR2 adsorption column (ensuring that the CR2 adsorption column is inserted into a collecting tube), centrifuging at 8000g/min for 1min, and discarding the liquid in the centrifuge tube;

F. adding 500 mu L of buffer GD into a CR2 adsorption column, centrifuging for 1 minute at 8000g/min, and discarding the liquid in a centrifuge tube; adding 600 microliter rinsing liquid PW into a CR2 adsorption column, centrifuging for 1 minute at 8000g/min, and discarding the liquid in a centrifuge tube;

G. adding 600 μ L of rinsing liquid PW into CR2 adsorption column, centrifuging at 8000g/min for 1min, and discarding the liquid in the centrifuge tube;

H. adding 500 microliter of absolute ethyl alcohol into a CR2 adsorption column, centrifuging for 1 minute at 8000g/min, and discarding the liquid in a centrifuge tube;

I. 12000g/min, centrifuging for 3 min, taking out the CR2 adsorption column, putting the CR2 adsorption column into a new 1.5mL enzyme-free centrifuge tube, standing for 3 min at room temperature, volatilizing absolute ethyl alcohol, and drying the adsorption film;

J. suspending and dripping 50 mu on the adsorption filmEnzyme-free ddH of L ₂ O (note that the adsorption membrane can not be contacted), covering a tube cover, standing for 5 minutes at room temperature, centrifuging for 1 minute at 12000g/min, discarding a CR2 adsorption column, and centrifuging the obtained liquid to obtain the extracted nucleic acid; storing in a low temperature refrigerator at-80 deg.C for use.

Example 4: multiplex qPCR assays

10 respiratory tract infectious samples (primary sequencing verified positive pathogens) were collected from hospitals, nucleic acid was extracted and detected by multiplex real-time fluorescent quantitative PCR technique, and the multiplex real-time fluorescent quantitative PCR technique was compared with bacterial culture and primary sequencing results, as shown in the following table:

the above table shows that the primers and probe sets for multiple respiratory infectious diseases pathogens designed by the invention are combined with the real-time fluorescent quantitative PCR technology, so that pathogens which cannot be detected by bacterial culture or first-generation sequencing can be detected, and the complementary effect on bacterial culture or first-generation sequencing results can be achieved; the established multiple real-time fluorescent quantitative PCR targeting 8 respiratory infectious disease pathogens has good application value.

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Claims

1. A multiplex qPCR detection primer combination for respiratory infectious disease pathogens, which is characterized in that: comprises specific primers and probes for detecting influenza A virus, influenza B virus, parainfluenza virus type 1, parainfluenza virus type 3, respiratory syncytial virus type A, rhinovirus, metapneumovirus and respiratory syncytial virus type B;

the specific primers are SEQ ID NO 1 and SEQ ID NO 2 for influenza A virus, SEQ ID NO 4 and SEQ ID NO 5 for influenza B virus, SEQ ID NO 7 and SEQ ID NO 8 for parainfluenza virus type 1, SEQ ID NO 10 and SEQ ID NO 11 for parainfluenza virus type 3, SEQ ID NO 13 and SEQ ID NO 14 for respiratory syncytial virus type A, SEQ ID NO 16 and SEQ ID NO 17 for rhinovirus, SEQ ID NO 19 and SEQ ID NO 20 for metapneumovirus, and SEQ ID NO 22 and SEQ ID NO 23 for respiratory syncytial virus type B;

the probe is SEQ ID NO. 3 for influenza A virus, SEQ ID NO. 6 for influenza B virus, SEQ ID NO. 9 for parainfluenza virus type 1, SEQ ID NO. 12 for parainfluenza virus type 3, SEQ ID NO. 15 for respiratory syncytial virus type A, SEQ ID NO. 18 for rhinovirus, SEQ ID NO. 21 for metapneumovirus, and SEQ ID NO. 24 for respiratory syncytial virus type B.

2. The multiplex qPCR detection primer combination for infectious respiratory disease pathogens according to claim 1 characterized in that: specific primers and probes of influenza A virus, influenza B virus, parainfluenza virus type 1 and parainfluenza virus type 3 are commonly used in detection; specific primers and probes of respiratory syncytial virus A, rhinovirus, metapneumovirus and respiratory syncytial virus B are commonly used in detection, and a human respiratory epithelial cell ribonuclease P gene is used as an internal reference.