Primer combination for simultaneously detecting enterovirus D68 and rhinovirus and application thereof

Abstract

The invention discloses a primer combination for simultaneously detecting enterovirus D68 and rhinovirus, belongs to the technical field of molecular biology, and aims to solve the problems of low single pathogen detection efficiency, remarkable cross reaction, HRV-C type missing detection and the like in the prior art. An HRV 5 'UTR conserved region and an EV-D68 VP1 gene hypervariable region are analyzed and screened as targets through bioinformatics; the nucleotide sequences of the specific primer aiming at the enterovirus D68 obtained by designing and screening are shown as SEQ ID NO: 1-SEQ ID NO: 2, and the nucleotide sequence of the probe is shown as SEQ ID NO: 3; the nucleotide sequences of the specific primer aiming at the rhinovirus are as shown in SEQ ID NO: 4-SEQ ID NO: 5, and the nucleotide sequence of the probe is as shown in SEQ ID NO: 6; the method is high in detection sensitivity and low in cost, and specificity verification shows that the method has no cross reaction with other genotypes of enteroviruses and common respiratory pathogens.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and particularly relates to primers for detecting enterovirus D68 and rhinovirus and their application in the preparation of a detection kit, and is particularly suitable for the differential diagnosis of pathogens of children with acute respiratory infection (ARI). Background Art

[0002] Acute respiratory infection (ARI) is one of the main causes of morbidity and hospitalization in children. Among them, enterovirus D68 (EV-D68) and rhinovirus (HRV) are the main pathogens causing this disease. Both EV-D68 and HRV belong to important pathogens of the genus Enterovirus in the family Picornaviridae and have a high clinical correlation. In the initial stage of both infections, they show non-specific respiratory symptoms (such as runny nose, cough, fever), and are often co-infected with other pathogens (such as respiratory syncytial virus), resulting in difficult clinical differentiation. It is worth noting that EV-D68 infection not only causes respiratory symptoms but is also closely related to neurological complications (such as acute flaccid myelitis). Although HRV infection is mainly mild, its high prevalence rate (accounting for 30%-50% of children with ARI cases), and type C (HRV-C) accounts for up to 46% in severely ill children, posing a challenge to public health management. Since there are essential differences in antiviral drug sensitivity and pathogenic characteristics among different genotypes, it is crucial to quickly and accurately distinguish EV-D68 and HRV infections for guiding individualized treatment and prevention and control strategies.

[0003] At present, the detection methods for enteroviruses mainly include virus isolation and culture, serological antibody detection, and PCR-based molecular diagnosis techniques. However, the traditional virus isolation method is time-consuming (3-7 days) and has low sensitivity; serological detection is easily interfered by cross-reactions. Although ordinary singleplex qPCR can improve sensitivity, it cannot achieve multi-target synchronous screening. Existing detection technologies mostly target a single pathogen (such as only detecting EV-D68 or HRV), and the two often coexist or are co-infected during the autumn and winter epidemic periods. If a step-by-step detection strategy is adopted (such as first detecting HRV and then detecting EV-D68 if negative), it will increase sample consumption, detection cost, and time delay. In addition, the enterovirus genome is highly variable, and it is difficult for existing multiplex PCR technologies to achieve broad-spectrum coverage of EV-D68 and HRV due to the complex design of primers and probes. For example, some HRV detection reagents cannot cover all genotypes (such as HRV-C) because they target genotype-specific regions (such as the VP4 / VP2 gene), and the 5'UTR conserved sequence has a high sequence similarity with the 5'UTR of enteroviruses such as EV-D68, which is prone to cross-reaction.

[0004] Therefore, there is an urgent need to develop a molecular diagnostic method with high sensitivity, strong specificity and multiplex detection ability to achieve an efficient discrimination scheme for the synchronous detection of HRV and EV-D68, providing technical support for precise diagnosis and treatment and epidemic control. Summary of the Invention

[0005] To solve the problems that existing detection methods cannot detect EV-E68 and HRV simultaneously and have insufficient coverage of genotypes, the present invention provides a primer combination for simultaneously detecting enterovirus D68 and rhinovirus. The nucleotide sequences of the specific primers for enterovirus D68 are shown as SEQ ID NO:1 - SEQ ID NO:2, and the nucleotide sequence of the probe is shown as SEQ ID NO:3; the nucleotide sequences of the specific primers for rhinovirus are shown as SEQ ID NO:4 - SEQ ID NO:5, and the nucleotide sequence of the probe is shown as SEQ ID NO:6. The primer combination of the present invention also includes the upstream primer, downstream primer, and probe of the internal standard gene ribonuclease P (RNase-P), and the nucleotide sequences are shown as SEQ ID NO:7 - 9.

[0006] The primer combination of the present invention mainly uses the multiplex fluorescence PCR analysis method to detect different pathogens by detecting different targets, so as to simultaneously detect and distinguish EV-D68 and HRV in a single-tube reaction system, providing a targeted strategy for subsequent treatment. The composition of the present invention has higher detection sensitivity, reaching 500 copies / mL, good specificity, and more accurate detection.

[0007] The fluorescent groups of the probes in the composition of the present invention are different from each other and do not interfere with each other.

[0008] In this article, "different from each other and do not interfere with each other" means that the fluorescent groups used for each probe in the composition are different and do not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX, and CY5 can be used. These groups have absorbance values that are not close, can select different channels, and thus do not interfere with each other.

[0009] The fluorescent reporter group of the EV-D68 detection probe is FAM; the fluorescent reporter group of the HRV detection probe is ROX; the fluorescent reporter group of the internal standard RNase-P detection probe is CY5; the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2, BHQ3.

[0010] Another object of the present invention is to apply the above primer combination in the preparation of a detection reagent or kit for simultaneously detecting enterovirus D68 and rhinovirus. The detection reagent or kit further includes other conventional reagents for multiplex fluorescence PCR, such as PCR reaction buffer, RT-PCR enzyme mixture, negative control product, and positive control product.

[0011] The negative control product is at least one of DEPC H 2 O and physiological saline; the positive control product is at least one of EV-D68, HRV, fragment nucleic acids of RNase-P, pseudovirus, etc.

[0012] The method for using the above multiplex fluorescence quantitative PCR detection reagent is as follows: (1) Extract or release the nucleic acid of the sample to be tested; (2) Perform fluorescence quantitative PCR on the nucleic acid obtained in step (1) using the above composition; (3) Obtain and analyze the results.

[0013] In the present invention, the sample for detection can be respiratory secretions such as throat swabs, nasal swabs, etc., but is not limited thereto.

[0014] The reaction conditions for the fluorescence quantitative PCR are as follows: Reverse transcription, temperature is 40 - 50 °C, time is 5 - 10 minutes, 1 cycle; pre-denaturation, temperature is 95 °C, time is 30 seconds - 1 minute, 1 cycle; denaturation, temperature is 95 °C, time is 5 - 20 seconds, annealing, temperature is 55 °C - 60 °C, time is 10 - 60 seconds, 30 - 50 cycles, and fluorescence is collected.

[0015] Advantages and technical effects of the present invention: (1) Based on the multiplex fluorescence PCR technology, the present invention has developed a primer combination for detecting EV-D68 and HRV. This primer combination has the advantages of convenience and rapidity, and can realize the simultaneous detection of two pathogens; (2) The method of the present invention has the advantages of high sensitivity, strong specificity, and high accuracy. The lowest detection limit for EV-D68 and HRV can reach 500 copies / mL; in addition, a positive control is set to participate in the whole process quality control of nucleic acid extraction, PCR amplification, etc., to avoid the generation of false positive or false negative results and improve the detection accuracy; (3) Compared with the step-by-step detection strategy, this method can simultaneously identify two pathogens in a single reaction, reduce the sample consumption by 50%, and shorten the detection time to 2 hours. It provides key technical support for the clinical rapid identification of EV-D68 and HRV infections, assessment of the risk of mixed infections, and guidance of antiviral treatment, and has significant clinical application value and public health prevention and control significance; The present invention has important clinical auxiliary value for the early diagnosis of children with acute respiratory tract infection and the observation of the disease course, so as to guide clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Results of detecting human rhinovirus and enterovirus D68 pseudovirus templates with different primer sets; Figure 2 Detection result diagram of the detection composition of the present invention; Figure 3 Results of HRV sensitivity detection; Figure 4 Results of EV-D68 sensitivity detection; Figure 5 Results of detecting clinical samples with enterovirus D68 and rhinovirus kits. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] The technical solutions of the present invention will be further described in detail below through examples. However, the content of the present invention is not limited thereto. In the following examples, the methods are conventional methods unless otherwise specified, and the materials, reagents, etc. are obtained from commercial sources or prepared according to conventional methods unless otherwise specified; Example 1: Design of primers and probes and synthesis of positive controls 1. Primer design and synthesis According to the HRV and EV-D68 genome sequences published in GeneBank, the relatively conserved regions were blasted using biological software Blast, and primers and probes were designed respectively for the 5'UTR of HRV and the VP1 region of EV-D68; in addition, primers and probes were designed for the internal reference gene ribonuclease P (RNase-P) for quality control; Table 1 The primers and probes used in the present invention are shown as follows: ; 2. Synthesis of positive controls In this example, the HRV, EV-D68 and RNase-P gene sequences were obtained by chemical synthesis and cloned and constructed into the pET-28b-MS2 vector, and armor RNA was prepared, concentrated and purified in Escherichia coli; the obtained armor RNA is an RNA nucleic acid sequence wrapped by the capsid protein of MS2 phage, which can be used as an internal reference control product for virus RNA nucleic acid extraction experiments and QPCR detection experiments; the preparation steps are as follows: (1)Entrusted Anhui General Biology to concatenate the detection target sequences of HRV, EV-D68, and RNase-P into the target gene (the sequence is shown in Table 2, SEQ ID NO:10), and insert it into the pET-28b-MS2 vector (prepared by the method in invention application CN 108795961 A). The phage MS2 (81-1740nt) region includes the 5' non-coding region sequence, maturase protein gene sequence, capsid protein gene sequence, packaging site, and replicase gene sequence; (2)Transfer the successfully constructed pET-28b-MS2 expression plasmid containing the detection targets of HRV, EV-D68, and RNase-P into the Escherichia coli prokaryotic expression strain BL21, induce expression with 0.5 mM IPTG for 8 h, centrifuge after ultrasonic disruption, and collect the precipitate to obtain armored RNA virus-like particles containing the detection targets of HRV, EV-D68, and RNase-P. The construction of the armored RNA virus-like particles was entrusted to Anhui General Biology to complete; Extract the RNA of the virus-like particles, measure the concentration by ultraviolet spectrophotometer, and calculate the copy number of RNA according to the detection target sequence and concentration. The calculation formula is as follows:

[0018] Table 2 Positive control detection target sequence 。

[0019] Example 2: Screening of primer-probe combinations for single target genes 1. Detection of human rhinovirus and enterovirus D68 pseudovirus templates with different primer sets (1)Preparation of the detection reaction system: The single RT-qPCR reaction system (20 μL) is as follows:

[0020] Note: In this example, the primers and probes used are the combinations in Table 1 of Example 1, and the template is the pseudovirus RNA containing HRV, EV-D68, and RNase-P (extracted from the armored RNA virus-like particles containing the detection targets of HRV, EV-D68, and RNase-P); (2)RT-qPCR reaction, the reaction program is as follows:

[0021] (3)Result interpretation: Screen the optimal primer combination according to the CT value and fluorescence intensity. The detection results are shown in Table 3 and Figure 1 as follows; Table 3. Detection results of human rhinovirus and enterovirus D68 pseudovirus templates with different primer sets

[0022] As can be seen from the detection results, when using the pseudovirus template as the template, both combination ① and combination ② can effectively detect rhinovirus RNA and EV-D68 RNA at different concentrations, and there is no significant difference in CT values.

[0023] 2. Detection of different types of human rhinovirus and EV-D68 by different primer sets Replace the pseudovirus template in the single RT-qPCR reaction system with HRV and EV-D68 nucleic acids of different genotypes. At the same time, set a negative control (NTC), and keep the other conditions unchanged. Detect the detection results of each primer set for HRV and EV-D68 nucleic acids of different genotypes. The detection results are shown in Table 4: Table 4 Detection results of different primer sets for different genotypes of HRV and EV-D68

[0024] As can be seen from the detection results, compared with HRV combination ①, HRV combination ② can detect more genotypes of HRV, and there is no significant difference in CT values; both combination ① and combination ② of EV-D68 can detect different genotypes of EV-D68, and combination ① has a lower CT value than combination ②.

[0025] 3. Cross-reaction verification of different primer sets for HRV and EV-D68 Select 12 other genotypes of enteroviruses and 8 common respiratory infection pathogens as cross-reaction pathogens. Replace the pseudovirus template in the single RT-qPCR reaction system with the corresponding cross-reaction pathogen nucleic acids, and keep the other conditions unchanged. Detect the specificity of each primer set; the detection results are shown in Table 5: Table 5 Detection results of cross-reaction verification of different primer sets for HRV and EV-D68

[0026] As can be seen from the detection results, HRV combination ① has a cross-reaction with coxsackievirus A2 and coxsackievirus A5, while HRV combination ② does not have a cross-reaction; neither EV-D68 combination ① nor EV-D68 combination ② has a cross-reaction.

[0027] Conclusion: After 3 rounds of screening tests, it can be seen from the experimental results of different primer sets for pseudovirus, different types of human rhinovirus, EV-D68 and cross-reaction evaluation that HRV combination ② has better amplification efficiency and no cross-reaction compared with HRV combination ①; while EV-D68 combination ① has better amplification efficiency. In summary, HRV combination ② and EV-D68 combination ① are the best primer sets.

[0028] Example 3: Preparation of Detection Kits for Enterovirus D68 and Rhinovirus and Establishment of Methods Using the best primer combinations (HRV combination ②, EV-D68 combination ①, and internal standard (ribonuclease P) gene primer set) screened in Example 2, a detection kit was prepared. The kit includes primer combinations, PCR reaction buffer, RT-PCR enzyme mixture, negative control product, and positive control product. The usage method of the detection reagents for enterovirus D68 and rhinovirus is as follows: (1) Preparation of the detection reaction system: The multiplex RT-qPCR reaction system (40 μL) is as follows:

[0029] Note: In this example, for HRV combination ②, EV-D68 combination ①, and the internal standard (ribonuclease P) gene primer set, the template is the pseudovirus RNA containing HRV, EV-D68, and RNase-P.

[0030] (2) RT-qPCR reaction, and the reaction procedure is as follows:

[0031] (3) Result analysis and determination:

[0032] The detection results are shown in Figure 2 As shown, both HRV and EV-D68 can be effectively amplified in the multiplex RT-qPCR reaction. The results indicate that the present invention has successfully established a detection method for enterovirus D68 and rhinovirus.

[0033] Example 4: Sensitivity Test of the Detection Method for Enterovirus D68 and Rhinovirus Take 200 μL of the armored RNA virus-like particles containing HRV, EV-D68, and RNase-P detection targets prepared in Example 1 to extract pseudovirus RNA. Measure the concentration by ultraviolet spectrophotometer, and calculate the copy number of RNA according to the detection target sequence and concentration. The calculation formula is as follows:

[0034] The obtained virus RNA template with a concentration of 5×10 6 copies / mL was serially diluted 10-fold to prepare pseudovirus RNA templates with concentrations of 5×10 6 copies / mL - 500 copies / mL for the sensitivity test of the detection method for enterovirus D68 and rhinovirus.

[0035] (1) Preparation of the detection reaction system: The multiplex RT-qPCR reaction system (40 μL) is as follows:

[0036] Note: In this example, for the HRV combination ② and EV-D68 combination, as well as the internal standard (ribonuclease P) gene primer set, the template is the pseudovirus RNA with a concentration of 5×10 6 copies / mL - 500 copies / mL.

[0037] (2) RT-qPCR reaction procedure:

[0038] The detection results are as Figure 3 , Figure 4 shown. The detection sensitivity of HRV and EV-D68 is 500 copies / mL.

[0039] Example 5: Interference resistance of the enterovirus D68 and rhinovirus detection kit The kit of Example 3 was analyzed for interfering substances by conventional methods. The experimental results showed that potential PCR inhibitors / interfering substances such as dexamethasone (50 μg / mL), cefmenoxime hydrochloride (50 μg / mL), zanamivir (100 μg / mL), ribavirin (100 μg / mL), clindamycin (50 μg / mL), tobramycin (50 μg / mL), heme (10 μg / mL), hemoglobin (10%) at certain concentrations had no obvious effect on this kit. The amplification results of the PCR reaction solution in the presence of infectious substances are shown in Table 6 below; Table 6 Results of the interference resistance test of the enterovirus D68 and rhinovirus detection kit .

[0040] Example 6: Clinical sample testing of the enterovirus D68 and rhinovirus detection kit Respiratory pharyngeal swab samples from 10 pediatric patients were collected, and the enterovirus D68 and rhinovirus detection kit in Example 3 was used for sample detection. The specific implementation steps are as follows: (1) Nucleic acid extraction: Take 200 μL of pharyngeal swab sample and use a viral DNA / RNA nucleic acid extraction kit to obtain viral RNA; (2) Preparation of the detection reaction system: The multiplex RT-qPCR reaction system (40 μL) is as follows:

[0041] (3) RT-qPCR reaction, reaction procedure:

[0042] (4)Result analysis and determination:

[0043] The detection results are as Figure 5 shown. Among the 10 children's throat swab samples, obvious amplification curves appeared in the internal standard CY5 channel, and the CT values ranged from 25 to 30; 2 cases of HRV virus were detected positive in the FAM channel; 2 cases of EV-D68 virus were detected positive in the ROX channel, and the results were verified by first-generation sequencing with an accuracy rate of 100%. The experimental results show that the enterovirus D68 and rhinovirus detection kit developed by the present invention has good detection performance in the detection of clinical samples and is a diagnostic technology with good prospects.

Claims

1. A primer combination for simultaneous detection of enterovirus D68 and rhinovirus, characterized in that: The nucleotide sequences of the specific primers for enterovirus D68 are shown in SEQ ID NO:1-SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3; the nucleotide sequences of the specific primers for rhinovirus are shown in SEQ ID NO:4-SEQ ID NO:5, and the nucleotide sequence of the probe is shown in SEQ ID NO:

6.

2. The primer combination for simultaneous detection of enterovirus D68 and rhinovirus according to claim 1, characterized in that: It also includes the nucleotide sequences of specific primers and probes of the internal reference gene ribonuclease P, as shown in SEQ ID NO:7-SEQ ID NO:

9.

3. Use of the primer combination described in claim 2 in the preparation of a detection reagent for simultaneously detecting enterovirus D68 and rhinovirus.

4. Use of the primer combination described in claim 2 in the preparation of a kit for simultaneously detecting enterovirus D68 and rhinovirus.

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