CN114990261B

CN114990261B - Multiplex qPCR detection reagent for detecting respiratory tract infectious disease pathogens

Info

Publication number: CN114990261B
Application number: CN202210612569.8A
Authority: CN
Inventors: 冯悦; 刘文锋; 刘丽; 夏雪山
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2022-06-01
Filing date: 2022-06-01
Publication date: 2024-05-07
Anticipated expiration: 2042-06-01
Also published as: CN114990261A

Abstract

The invention discloses a multiplex qPCR detection reagent for detecting respiratory tract infectious disease pathogens, which comprises specific primers and probes for detecting EB virus, haemophilus influenzae, mycoplasma pneumoniae, streptococcus pneumoniae, moraxella catarrhalis, cytomegalovirus, respiratory syncytial virus, rhinovirus and influenza B virus; the invention detects the pathogen target gene by using a real-time fluorescent quantitative PCR technology; 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 pathogens of respiratory tract infectious diseases, and has important significance for clinical early molecular diagnosis of respiratory tract infection patients.

Description

Multiplex qPCR detection reagent for detecting respiratory tract infectious disease pathogens

Technical Field

The invention belongs to the technical field of biology, and relates to a specific primer and a probe for detecting 9 respiratory tract infectious disease pathogens.

Background

Pathogenic microorganisms invade the respiratory tract to reproduce and induce a disease called respiratory tract infection (Respiratory Tract Infection, RTI). The respiratory tract infection can cause clinical symptoms such as common cold, acute viral pharyngitis, laryngitis, acute bronchitis, acute bronchiolitis, pneumonia and the like; meanwhile, respiratory tract infections are a major fatal disease. Worldwide, acute lower respiratory tract infections are the leading cause of death in children under 5 years of age, far above other causes of death such as diarrhea and accidental asphyxia. A variety of pathogenic microorganisms such as viruses, bacteria, fungi, mycoplasma, chlamydia, and the like can cause respiratory tract infections.

Clinically, there are multiple viruses including respiratory syncytial virus (Respiratory syncytial virus, RSV), influenza virus (IFV), rhinovirus (Human rhinovirus, HRV), cytomegalovirus (Human cytomegalovirus, HCMV), epstein-Barr virus (EBV), and the like. While bacteria concentrate on Streptococcus pneumoniae (Streptococcus pneumoniae, SP), haemophilus influenzae (Haemophilus influenzae, HI), moraxella catarrhalis (Moraxella catarrhalis, MC), and the like. In addition, mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) and the like can cause respiratory tract infections as well.

Early, timely and accurate diagnosis is helpful for clinical correct and reasonable medication, helps patients to recover health as early as possible, and reduces the death rate. The bacterial and virus culture separation method is one of the clinical pathogen detection methods, has low detection rate and long time consumption, and can not meet the detection requirements of a large number of clinical samples; the NGS sequencing technology is mainly applied to the detection of atypical pathogens, and has the defects of high cost, long time consumption, easy pollution and the like at present; immunological methods based on the principle of antigen-antibody reaction are prone to false positives due to cross reaction; the gene chip technology and the digital PCR technology are emerging molecular biology detection methods, and face the problems of higher cost, easy pollution, standardized production and the like; the primer design requirement of the loop-mediated isothermal amplification technology is high, and high-flux detection of various pathogens is difficult to realize; although the PCR combined with Sanger sequencing technology has high specificity, the detection is time-consuming, the flux is low, and the PCR combined with Sanger sequencing technology is not suitable for clinical immediate diagnosis.

The Real-time fluorescent quantitative PCR (Real-time fluoroscopy quantitative PCR, qPCR) technology was first marketed by the United states ABI (Applied Biosystems) company, and has been widely used in rapid progress over the last 20 years. The multiplex fluorescent quantitative PCR technology has the advantages of strong specificity, high sensitivity, simple operation, short time, low cost, suitability for screening a large number of samples of people and the like in the aspect of respiratory tract infectious disease pathogen physical examination.

Disclosure of Invention

The invention provides a multiplex qPCR detection reagent for detecting respiratory tract infectious disease pathogens by using a targeted pathogen genome conserved region amplification and multiplex real-time fluorescent quantitative PCR technology, which comprises specific primers and probes for detecting 9 respiratory tract infectious disease pathogens and further comprises other conventional detection reagents for multiplex qPCR detection.

The multiple qPCR detection reagent comprises specific primers and probes for detecting EB virus, haemophilus influenzae, mycoplasma pneumoniae, streptococcus pneumoniae, moraxella catarrhalis, cytomegalovirus, respiratory syncytial virus, rhinovirus and influenza B virus; wherein the specific primers are SEQ ID NO. 1 and SEQ ID NO. 2 for EB virus, SEQ ID NO. 4 and SEQ ID NO. 5 for Haemophilus influenzae, SEQ ID NO. 7 and SEQ ID NO. 8 for Mycoplasma pneumoniae, SEQ ID NO. 13 and SEQ ID NO. 14 for Streptococcus pneumoniae, SEQ ID NO. 16 and SEQ ID NO. 17 for Moraxella catarrhalis, SEQ ID NO. 19 and SEQ ID NO. 20 for cytomegalovirus, SEQ ID NO. 22 and SEQ ID NO. 23 for respiratory syncytial virus, SEQ ID NO. 25 and SEQ ID NO. 26 for influenza B virus, SEQ ID NO. 28 and SEQ ID NO. 29 for rhinovirus; the probes are SEQ ID NO. 3 for EB virus, SEQ ID NO. 6 for haemophilus influenzae, SEQ ID NO. 9 for mycoplasma pneumoniae, SEQ ID NO. 15 for streptococcus pneumoniae, SEQ ID NO. 18 for Moraxella catarrhalis, SEQ ID NO. 21 for cytomegalovirus, SEQ ID NO. 24 for respiratory syncytial virus, SEQ ID NO. 27 for influenza B virus and SEQ ID NO. 30 for rhinovirus.

The method for using the multiplex qPCR detection reagent comprises the following steps:

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

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

B. placing 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. And (3) taking 700 mu L of lysate in the step B, and performing nucleic acid extraction by using a Rhizopus 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 for targeting 9 pathogens through multiplex real-time fluorescent quantitative PCR, and judging the result according to a Ct value by using the ribonuclease P gene of the human airway epithelial cells as an internal reference;

Specific primers and probes for detecting EB virus, haemophilus influenzae and mycoplasma pneumoniae are used together in detection; specific primers and probes for detecting streptococcus pneumoniae, moraxella catarrhalis and cytomegalovirus are used together in detection, and specific primers and probes for detecting respiratory syncytial virus, rhinovirus and influenza B virus are used together in detection, with the human respiratory epithelial cell ribonuclease P gene as an internal reference;

The nucleotide sequences of specific primers and probes for detecting 9 pathogens and reference genes are shown as SEQ ID NO. 1-SEQ ID NO. 30;

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

(1) The DNA reaction system was 40. Mu.L

Amplification conditions: 95 ℃ for 30s; collecting fluorescence at 95 ℃,5s and 58 ℃ for 30s, and 45 cycles;

(2) The RNA reaction system was 40. Mu.L

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

3. The judging of the detection result comprises the following steps:

(1) The Ct value of the internal reference (ribonuclease P gene and RNP gene of the human airway epithelial cells) is less than or equal to 36, and the negative control group and the template-free control group have no Ct value; if the detection is not consistent with the requirement, carrying out multiplex real-time fluorescent quantitative PCR detection again, or extracting nucleic acid again to carry out multiplex real-time fluorescent quantitative PCR detection;

(2) The Ct value of the pathogen is less than or equal to 36.0, and if the Ct value is more than 36.0, single real-time fluorescence quantitative PCR verification is required for the pathogen;

(3) The amplification curve is standard "S" and free of abnormal fluctuations.

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

1. the primer and probe set for detecting 9 respiratory tract infectious disease pathogens provided by the invention has the advantages of high detection efficiency and accurate detection result, and can rapidly complete pathogen diagnosis with low cost;

2. the invention carries out qualitative detection on pathogen specific target genes on the basis of a real-time fluorescent quantitative PCR technical platform, can detect a plurality of samples by 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 showing the amplification of EB virus by single fluorescent quantitative PCR;

FIG. 2 is a graph of the single fluorescent quantitative PCR amplification of Haemophilus influenzae;

FIG. 3 is a graph of single fluorescent quantitative PCR amplification of mycoplasma pneumoniae;

FIG. 4 is a graph showing the single fluorescent quantitative PCR amplification of the internal reference RNP gene;

FIG. 5 is a multiplex real-time fluorescent quantitative PCR amplification graph of EB virus, haemophilus influenzae, mycoplasma pneumoniae and internal reference RNP genes;

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

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

FIG. 8 is a graph of single fluorescent quantitative PCR amplification of cytomegalovirus;

FIG. 9 is a graph of multiplex fluorescence quantitative PCR amplification of Streptococcus pneumoniae, moraxella catarrhalis, cytomegalovirus;

FIG. 10 is a graph of single fluorescent quantitative PCR amplification of respiratory syncytial virus;

FIG. 11 is a graph of single fluorescent quantitative PCR amplification of influenza B virus;

FIG. 12 is a plot of single fluorescent quantitative PCR amplification of rhinoviruses;

FIG. 13 is a graph of multiplex fluorescence quantitative PCR amplification of respiratory syncytial virus, influenza B virus, rhinovirus;

FIG. 14 shows the experimental results of detection sensitivity of EB virus using multiplex real-time fluorescent quantitative PCR amplification system;

FIG. 15 shows the experimental results of the detection sensitivity of Haemophilus influenzae using a multiplex real-time fluorescent quantitative PCR amplification system;

FIG. 16 shows the experimental results of detection sensitivity of Mycoplasma pneumoniae by using a multiplex real-time fluorescent quantitative PCR amplification system;

FIG. 17 shows the experimental results of the detection sensitivity of the internal reference RNP gene by using a multiplex real-time fluorescent quantitative PCR amplification system.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described by the following specific embodiments, but the invention is not limited to the examples.

The materials used in the examples below are not limited to the above list, but may be replaced with other similar materials, and the apparatus is not specified, and the person skilled in the art should be aware of the use of conventional materials and apparatus according to conventional conditions, or according to conditions suggested by the manufacturer.

Example 1: design of specific primers and probes

A. The pathogen gene reference sequences were downloaded in the NCBI (National Center for Biotechnology Information ) website as follows: EB virus Virion glycoprotein gL encoding gene, haemophilus influenzae Hypothetical protein encoding gene, mycoplasma pneumoniae Toxin encoding gene, streptococcus pneumoniae INTERMEDILYSIN encoding gene, moraxe catarrhalis Membrane protein encoding gene, cytomegalovirus Regulatory protein IE encoding gene, respiratory syncytial virus Matrix protein encoding gene, influenza B virus Matrix protein 1 encoding gene, rhinovirus Polyprotein encoding gene 20 each;

B. Nucleotide sequences were aligned 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 ℃;

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

(3) Primer dimer is not generated, and the hairpin structure software evaluation result is OK;

(4) Amplified fragment sizes are generally less than 200bp;

C. primer and probe BLAST evaluation: the primer probe nucleotide sequence which is designed preliminarily is compared again by using the BLAST retrieval function in NCBI website, and the primer and probe sequence with high specificity are selected;

The nucleotide sequences of specific primers and probes for targeting 9 respiratory tract infectious disease pathogens and internal reference RNP genes are shown in SEQ ID NO. 1-SEQ ID NO. 30, and the following table is provided;

。

Example 2: real-time fluorescent quantitative PCR method establishment

1. Plasmid Synthesis

To evaluate pathogen primers, probe specificity, sensitivity, and method stability, commercial companies (Zhongmeitai and organism, beijing) were commissioned to synthesize plasmids based on primer targeting nucleotide sequences, and the nucleotide sequences linked to the plasmids were shown as SEQ ID NO: 31-SEQ ID NO: 40.

2. Specificity, sensitivity and repeatability experiments

(1) Specificity (specificity)

The single real-time fluorescent quantitative PCR is carried out, and the reaction system and conditions are as follows:

carrying out a single real-time fluorescent quantitative PCR experiment by taking a plasmid as a template, and configuring and setting the reaction system as follows:

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

Taking a synthesized plasmid standard as a positive substance, performing a single fluorescent quantitative PCR experiment (a reaction system and amplification conditions are as follows) by using a Ai Kerui biological company Pro Taq HS premixed probe method qPCR kit, and simultaneously detecting plasmid mixed samples of the 9 respiratory pathogens and 1 internal reference RNP genes (the gradient dilution concentration is 10 ⁵copies/μL-10³ copies/. Mu.L); the results of pathogen single fluorescent quantitative PCR experiments are shown in figures 1-4, 6-8 and 10-12, and the amplification results show that the 9 pathogens and the internal reference RNP genes have amplification curves, and no nonspecific amplification exists, so that the designed primers and probes have good specificity.

Multiplex real-time fluorescent quantitative PCR, reaction system and conditions were as follows:

Amplification conditions: 30sec at 95 ℃; (fluorescence was collected at 95℃for 5sec and 58℃for 30 sec) (45 cycles;

Taking the synthesized plasmid standard as a positive substance, and simultaneously detecting plasmid mixed samples (the gradient dilution concentration is 10 ⁵copies/μL-10³ copies/. Mu.L) of the 9 respiratory pathogens and 1 internal reference RNP genes; the results of pathogen multiplex fluorescence quantitative PCR experiments (the reaction system and the amplification conditions are as follows) are shown in figures 5, 9 and 13, and the amplification results show that the 9 pathogens and the internal reference RNP genes have amplification curves, and the primer and the probe cannot influence each other after being combined.

(2) Sensitivity of

The method of multiplex qPCR detects plasmid templates of 10⁷、10⁶、10⁵、10⁴、10³、10²、10¹、10⁰copies/μL orders of magnitude diluted by serial concentration gradients, and determines the lowest plasmid copy value which can be detected by the method of multiplex qPCR detection; as shown in FIGS. 14-17, the detection lower limit of EB virus, haemophilus influenzae, mycoplasma pneumoniae and internal reference RNP can all reach the order of 10 ¹ opies/. Mu.L, and the detection sensitivity of the other 6 pathogens can all reach the order of 10 ¹ opies/. Mu.L.

(3) Repeatability of

To verify the stability of the established multiplex qPCR assay, experiments were performed with plasmids on the order of 10 ³ copies/. Mu.L, and batch-to-batch reproducibility experiments were performed, respectively; the plasmids were subjected to fluorescent quantitative PCR experiments with specific primer and probe sets for each pathogen group, CV values were calculated for intra-batch and inter-batch experiments for three consecutive weeks, and the reproducibility results are shown in the following table:

According to the repeated experimental results, the established multiplex real-time fluorescence quantification method based on 9 respiratory tract infectious disease pathogens and 1 reference gene has higher stability (CV is less than 5%).

Example 3: sample nucleic acid (DNA & RNA) extraction

The DNA/RNA extraction of throat or nasal swabs from patients with respiratory tract infections was performed in this example using a commercial TIANamp Virus DNA/RNA Kit (TIANGEN, beijing) and the detailed extraction method was as follows:

A. Preparation of CARRIER RNA aqueous solution: absorbing 310 mu L of enzyme-free ddH ₂ O to dissolve 310 mu G CARRIER RNA dry powder, sufficiently shaking and then briefly centrifuging to obtain CARRIER RNA aqueous solution with the final concentration of 1 ng/mu L, and preserving at low temperature for later use;

B. CARRIER RNA preparation of working solution: according to the number (n) of the extracted samples, adding buffer solution GB, CARRIER RNA aqueous solution and proteinase K with the volumes of 0.22mL multiplied by n,6.16 mu L multiplied by n and 20 mu L multiplied by n respectively, shaking and fully mixing, and centrifuging for short to collect wall-hanging liquid;

C. Sample extraction pretreatment: the indoor quality control product is liquid, the liquid does not need to be treated, and the inactivated strain and the strain are diluted by adding 1mL of physiological saline; 200 mu L of the uniformly mixed sample and 220 mu L of CARRIER RNA working solution are sucked, a 1.5mL enzyme-free centrifuge tube is added, and after the mixture is fully mixed by shaking, the wall-hanging liquid is collected by brief centrifugation;

D. incubation: placing the mixed liquid into a water bath kettle with the temperature of 56 ℃, incubating for 15 minutes, and manually shaking and uniformly mixing every 5 minutes during the incubation; the wiping paper sucks water on the outer wall of the centrifuge tube, and the wall-mounted liquid is collected in a short centrifugation mode; sucking 0.25mL of absolute ethyl alcohol, adding the absolute ethyl alcohol into a centrifuge tube, oscillating for 15 seconds, fully and uniformly mixing, standing for 5 minutes at room temperature, and centrifuging for a short time to collect wall-mounted liquid;

E. And (3) transferring: transferring all the liquid in the centrifuge tube into a CR2 adsorption column (ensuring that the CR2 adsorption column is inserted into a collecting pipe), centrifuging for 1min at 8000 Xg/min, 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 the centrifuge tube; adding 600 mu L of rinsing solution PW into the CR2 adsorption column, centrifuging for 1 min at 8000g/min, and discarding the liquid in the centrifuge tube;

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

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

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

J. Suspending and dripping 50 μl of enzyme-free ddH ₂ O (note that the adsorption membrane cannot be contacted) onto the adsorption membrane, covering a tube cover, standing at room temperature for 5min, centrifuging at 12000g/min for 1 min, discarding CR2 adsorption column, and centrifuging to obtain liquid, namely extracted nucleic acid; placing in a refrigerator at-80deg.C, and storing.

Example 4: multiplex qPCR detection

A. DNA pathogen detection:

The reaction system of group 1 is as follows, the reaction system is 40. Mu.L, the combination of the primer and the probe is as follows in group 1:

the reaction system of group 2 is as follows, the reaction system is 40 mu L, and the combination of the primer and the probe is as shown in group 2:

；

Amplification conditions: 30sec at 95 ℃; (fluorescence was collected at 95℃for 5sec and 58℃for 30 sec) (45 cycles).

2. RNA pathogen detection

The reaction system was 40. Mu.L, and the combination of primers and probes was set forth in group 3:

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

In this example, the extracted nucleic acid was subjected to multiplex qPCR detection under the above conditions using a commercial one-step fluorescent quantification kit (nuback, nanjing) from nuback biosystems.

Comparing the results of 5 cases of respiratory tract infection samples detected by the multiplex real-time quantitative PCR technique with the results of bacterial culture or first-generation sequencing, wherein the results are shown in the following table;

As can be seen from the table, the pathogen which can not be detected by bacterial culture or first-generation sequencing can be detected by combining the primer and the probe set for the multiple respiratory tract infectious disease pathogens designed by the invention with the real-time fluorescent quantitative PCR technology, and the result of bacterial culture or first-generation sequencing can be supplemented; the established multiplex real-time fluorescent quantitative PCR targeting 9 respiratory tract infectious disease pathogens and 1 human respiratory tract epithelial cell ribonuclease P gene has good application value.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

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Claims

1. A multiplex qPCR detection reagent for detecting pathogens of respiratory tract infectious diseases, characterized in that: specific primers and probes for detecting EB virus, haemophilus influenzae, mycoplasma pneumoniae, streptococcus pneumoniae, moraxella catarrhalis, cytomegalovirus, respiratory syncytial virus, rhinovirus, influenza B virus;

The specific primers are SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 13 and SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 17, SEQ ID NO. 19 and SEQ ID NO. 20, SEQ ID NO. 22 and SEQ ID NO. 23, SEQ ID NO. 25 and SEQ ID NO. 26, SEQ ID NO. 28 and SEQ ID NO. 29, respectively, for EB virus, for haemophilus influenzae, for mycoplasma pneumoniae, for streptococcus pneumoniae, for Moraxella catarrhalis, for respiratory syncytial virus;

The probes are SEQ ID NO. 3 for EB virus, SEQ ID NO. 6 for haemophilus influenzae, SEQ ID NO. 9 for mycoplasma pneumoniae, SEQ ID NO. 15 for streptococcus pneumoniae, SEQ ID NO. 18 for Moraxella catarrhalis, SEQ ID NO. 21 for cytomegalovirus, SEQ ID NO. 24 for respiratory syncytial virus, SEQ ID NO. 27 for influenza B virus and SEQ ID NO. 30 for rhinovirus.

2. The multiplex qPCR detection reagent for detecting pathogens of respiratory tract infectious diseases according to claim 1, wherein: specific primers and probes for detecting EB virus, haemophilus influenzae and mycoplasma pneumoniae are used together in detection; specific primers and probes for detecting streptococcus pneumoniae, moraxella catarrhalis and cytomegalovirus are used together in detection, and specific primers and probes for detecting respiratory syncytial virus, rhinovirus and influenza B virus are used together in detection, with the human respiratory epithelial cell ribonuclease P gene as an internal reference;

Specific primers of human airway epithelial cell ribonuclease P gene are SEQ ID NO. 10 and SEQ ID NO. 11, and probe is SEQ ID NO. 12.