CN111088408A - Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus - Google Patents

Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus Download PDF

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CN111088408A
CN111088408A CN202010202863.2A CN202010202863A CN111088408A CN 111088408 A CN111088408 A CN 111088408A CN 202010202863 A CN202010202863 A CN 202010202863A CN 111088408 A CN111088408 A CN 111088408A
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朱娟娟
李烈军
谢龙旭
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Guangzhou Hybribio Medical Laboratory Co ltd
Hybribio Ltd
Guangzhou Kaipu Pharmaceutical Technology Co ltd
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Abstract

The invention discloses a detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus. The invention combines the design of a primer probe and optimization exploration of a compatible detection system, develops a group of novel coronavirus (2019-nCoV/SARS-CoV-2), influenza A virus (FluA), influenza B virus (FluB) and Respiratory Syncytial Virus (RSV) nucleic acid combined detection primer and probe combination and a combined nucleic acid detection kit, wherein the sequences of the primer and the probe are shown as SEQ ID NO: 1-18. The kit has the advantages of high detection sensitivity, good repeatability, and low false negative and false positive, and can effectively identify respiratory tract infection caused by new coronavirus infection and common viral influenza and respiratory syncytial virus, thereby realizing accurate diagnosis and subsequent accurate treatment of patients.

Description

Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus
Technical Field
The present invention belongs to the field of biomedicine and clinical diagnosis technology. More particularly, relates to a novel nucleic acid combined detection kit for coronavirus (2019-nCoV/SARS-CoV-2), influenza A virus (FluA), influenza B virus (FluB) and Respiratory Syncytial Virus (RSV).
Background
Since 12 months in 2019, the novel coronavirus 2019-nCoV/SARS-CoV-2 is spreading in the world, but human beings lack effective antiviral vaccines and specific drugs, and the accurate detection and diagnosis of infectors or carriers are of great importance to the control of epidemic situations. In addition, the clinical manifestations of the infection of various related viruses such as influenza a virus, influenza b virus, respiratory syncytial virus and the like in the high-incidence stage are similar to those of the novel coronavirus pneumonia, and the clinical manifestations and the chest imaging identification are difficult. Furthermore, previous studies showed that both new coronavirus infected and non-infected patients had a co-infection (as reported in 8274 clinical diagnoses of novel coronavirus in Wuhan City). If fever cases can not be diagnosed quickly and accurately in time, influenza cases are likely to be treated by being infected by the novel coronavirus, patients have more combined infection and large cross infection hidden danger, and the prevention and control of the novel coronavirus are affected extremely adversely.
Therefore, clinical diagnostic tools need to be improved, and screening of other common respiratory pathogens besides the new coronavirus should be highly appreciated. During the course of collecting new or suspected patients with coronary pneumonia, the possibility of other infections needs to be assessed in order to make clinical classification, isolation and treatment timely.
At present, the first line of clinical practice urgently needs to quickly identify related products of other respiratory viruses such as new coronavirus, influenza virus and the like, provides accurate detection for clinical practice, is beneficial to selecting a reasonable treatment scheme, greatly lightens the burden of a hospital, and enables precious treatment resources to be intensively used for treating patients with the new coronavirus.
Disclosure of Invention
The invention aims to overcome the defects of the existing technology for differential detection and diagnosis of other respiratory viruses such as new coronavirus, influenza virus and the like, and provides a novel nucleic acid joint detection kit for coronavirus (2019-nCoV), influenza A virus (FluA), influenza B virus (FluB) and Respiratory Syncytial Virus (RSV). The kit has high detection sensitivity, good repeatability and low false negative and false positive, and can effectively identify respiratory tract infection caused by new coronavirus infection, common viral influenza and respiratory syncytial virus, thereby realizing accurate diagnosis and subsequent accurate treatment of patients.
The invention aims to provide a group of novel nucleic acid joint detection primers and probe combinations for coronavirus, influenza A virus, influenza B virus and respiratory syncytial virus.
The invention also aims to provide a novel nucleic acid joint detection kit for coronavirus, influenza A virus, influenza B virus and respiratory syncytial virus.
The above purpose of the invention is realized by the following technical scheme:
a group of novel nucleic acid joint detection primers and probe combinations for coronavirus, influenza A virus, influenza B virus and respiratory syncytial virus comprise the following components:
(1) the primer pair and the probe for detecting the 2019-nCoV ORF1ab (open reading frame, ORF1 ab) target gene have nucleotide sequences shown in sequence as SEQ ID NO: 1-3;
(2) the nucleotide sequence of the primer pair and the probe for detecting the 2019-nCoV nucleocapsid protein N (nucleoprotein, N) target gene is shown as SEQ ID NO: 4-6;
(3) the nucleotide sequences of the primer pair and the probe for detecting the target gene of the influenza A virus are shown as SEQ ID NO: 7-9;
(4) the nucleotide sequences of the primer pair and the probe for detecting the target gene of the influenza B virus are shown as SEQ ID NO: 10-11 and SEQ ID NO: 12 is shown in the specification;
(5) the nucleotide sequences of the primer pair and the probe for detecting the target gene of the respiratory syncytial virus are shown as SEQ ID NO: 13-15;
(6) the nucleotide sequences of the primer pair and the probe for detecting the internal standard B2M are shown as SEQ ID NO: 16-18.
Specifically, primer and probe sequences are shown in table 1.
Preferably, two ends of the probe are respectively marked with one of dyes with the maximum emission wavelength of 518nm, 538 nm-553 nm, 574 nm-575 nm, 607 nm-615 nm, 640nm, 666 nm-667 nm and 690nm, and the dyes are different.
More preferably, the probe is labeled at the 5 'end with ROX, FAM, HEX or CY5 fluorogenic group and at the 3' end with MGB.
Most preferably, SEQ ID NO: 3 and SEQ ID NO: 6, marking a ROX fluorescence generating group at the 5 'end of the probe and marking MGB at the 3' end;
SEQ ID NO: 9 and SEQ ID NO: 12, the 5 'end of the probe is marked with FAM fluorescence generating group, and the 3' end is marked with MGB
SEQ ID NO: 15, marking a HEX fluorescence generating group at the 5 'end of the probe shown in the figure, and marking MGB at the 3' end;
SEQ ID NO: 18 is labeled with CY5 fluorophore at the 5 'end and MGB at the 3' end.
The primer and probe combination developed by the invention can effectively identify new coronavirus, common influenza A and B virus and respiratory syncytial virus in the same tube reaction, and has the advantages of high detection sensitivity, good repeatability, low false negative and low false positive.
The invention constructs a novel nucleic acid joint detection kit for coronavirus, influenza A virus, influenza B virus and respiratory syncytial virus based on the primer, and the kit contains the primer and a probe combination.
Preferably, the concentration of the probe used for detecting the target gene is 180-220nM, the concentration of the probe used for detecting the internal standard is 80-120 nM, the concentration of the upstream and downstream primers used for detecting the target gene is 180-220nM, and the concentration of the primer used for detecting the internal standard is 80-120 nM.
More preferably, the target gene is detected using a probe at a concentration of 200 nM, the internal standard is detected using a probe at a concentration of 100nM, the upstream and downstream primers for the target gene are detected using a concentration of 200 nM, and the internal standard is detected using a concentration of 100 nM.
As an alternative preferred scheme, the combined detection kit also comprises a PCR buffer solution, an enzyme mixed solution, a blank control and a positive control.
Preferably, the PCR buffer consists of: 26-30mol/L pH8-8.5 Tris-HCl, 15-25mmol/L (NH)4)2SO4，28-32mmol/L KC1，3.5-4.5mmol/ L MgCl2，0.15-0.25mol/ L dNTPs。
More preferably, the PCR buffer consists of: 28mol/L Tris-HCl pH8.0, 20mmol/L (NH)4)2SO4，30mmol/L KC1，4.0mmol/ L MgCl2, 0.2mol/ L dNTPs。
Preferably, the enzyme mixture comprises hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG); wherein the dosage of the hot start Taq enzyme is 3-8U/person, the dosage of the reverse transcriptase is 3-8U/person, and the dosage of the uracil glycosylase is 0.05-0.2U/person.
Preferably, the blank is rnase-free water;
preferably, the positive control: 2019-nCoV ORF1ab gene plasmid with concentration of 1.0X 105copies/ml; 2019-nCoVN gene plasmid with the concentration of 1.0 multiplied by 105copies/ml; influenza A virus plasmid at a concentration of 1.0X 105copies/ml; influenza B virus plasmid at a concentration of 1.0X 105copies/ml; respiratory syncytial virus plasmid at a concentration of 1.0X 105copies/ml; internal standard plasmid at a concentration of 1.0X 104copies/ml。
According to the kit developed by the invention, the human B2M protein gene is selected as the endogenous internal standard for the internal standard, the whole experimental process of sample collection, extraction and detection is monitored, and false negative is avoided. Meanwhile, UNG enzyme + dUTP anti-pollution measures are set, so that false positive results caused by PCR product pollution are avoided.
The invention provides a kit which is based on a multiple fluorescence PCR technology and can detect a first group of influenza A (FluA) viruses, a second group of influenza B (FluB) viruses, a third group of new coronavirus 2019-nCoV ORF1ab gene and N gene, a fourth group of respiratory syncytial viruses and a fifth group of B2M gene of intracellular standards in the same reaction tube by performing synchronous nucleic acid amplification and detection through a real-time fluorescence PCR instrument. And the fifth group synchronously detects the intracellular conserved gene B2M, and is used for evaluating the quality of the collected sample and PCR inhibition factors and controlling the quality of the detection process. The Tm of all the probes of the invention is nearly the same and about 8-10 ℃ higher than the Tm of the primers, and the Tm of all the primers is nearly the same at 58-60 ℃, so that the probes are ensured to keep combining with the target sequence when the primers are extended.
The kit can classify four types of viruses including new coronavirus 2019-nCoV, influenza A (FluA), influenza B (FluB) and respiratory syncytial virus, and can also classify the viruses through internal classificationSign boardThe quality control is carried out on the detection process, and UNG enzyme + dUTP anti-pollution measures are set, so that false positive results caused by PCR product pollution are avoided.
The invention has the following beneficial effects:
the invention develops a group of novel coronavirus (2019-nCoV), influenza A virus (FluA), influenza B virus (FluB) and Respiratory Syncytial Virus (RSV) nucleic acid combined detection primers and probe combinations, can effectively identify the novel coronavirus, the common influenza A virus, the influenza B virus and the respiratory syncytial virus in the same tube reaction, and has the advantages of high detection sensitivity, good repeatability, low false negative and low false positive.
Meanwhile, a novel coronavirus (2019-nCoV), influenza A virus (FluA), influenza B virus (FluB) and Respiratory Syncytial Virus (RSV) nucleic acid combined detection kit is also constructed, a primer probe combination and a corresponding detection system are obtained through optimization and exploration, the kit detection comprises the novel coronavirus 2019-nCoV (ROX channel), the influenza A/B virus (FAM channel), the respiratory syncytial virus (HEX channel) and an endogenous internal standard (CY 5 channel), and the human RNA internal standard B2M is used as an internal reference for monitoring sample collection, extraction and amplification, namely the whole detection process, so that a false negative result is avoided; setting UNG enzyme + dUTP anti-pollution measures to avoid false positive results caused by PCR product pollution; and the whole detection process can be completed within 2 hours by matching with a full-automatic nucleic acid extraction platform.
The kit has high detection sensitivity, good repeatability and low false negative and false positive, can effectively identify respiratory tract infection caused by new coronavirus infection and common viral influenza and respiratory syncytial virus, thereby realizing accurate diagnosis and subsequent accurate treatment of patients, providing accurate detection for quickly identifying other respiratory tract viruses such as new coronavirus, influenza virus and the like at the first line of clinic, being beneficial to selecting a reasonable treatment scheme, greatly reducing the burden of hospitals, leading precious treatment resources to be intensively used for treating the patients with the new coronavirus, and having important application value for controlling the epidemic situation.
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FIG. 1 shows the detection of different concentrations (1X 10) of the A.fluid quality control substance in accordance with the present invention1copies/ml、1×102copies/ml、1×103copies/ml、1×104copies/ml、1×105copies/ml、1×106copies/ml).
FIG. 2 shows the detection of different concentrations (1X 10) of the quality control material of the second fluid according to the present invention1copies/ml、1×102copies/ml、1×103copies/ml、1×104copies/ml、1×105copies/ml、1×106copies/ml).
FIG. 3 shows the detection of different concentrations (1X 10) of quality control material for respiratory syncytial virus according to the present invention1copies/ml、1×102copies/ml、1×103copies/ml、1×104copies/ml、1×105copies/ml、1×106copies/ml).
FIG. 4 shows the detection of different concentrations (1X 10) of the novel crown quality control substance in the invention1copies/ml、1×102copies/ml、1×103copies/ml、1×104copies/ml、1×105copies/ml、1×106copies/ml).
FIG. 5 is an internal assay of the inventionQuality control product with different concentrations (1 × 10)1copies/ml、1×102copies/ml、1×103copies/ml、1×104copies/ml、1×105copies/ml、1×106copies/ml).
FIG. 6 shows that the Guangdong CDC novel coronal mesenchyme evaluation control substance 5330 of the present invention is diluted at different concentrations (1 × 10)1copies/ml、1×103copies/ml、1×104copies/ml、1×105copies/ml).
FIGS. 7-24 are graphs showing the amplification of 18 samples of quality control products from Bandwidth corporation, third party, in accordance with the present invention.
FIG. 25 shows the amplification results of positive control and blank control in the detection kit of the present invention.
FIG. 26 is a graph showing the amplification of interventricular quality control samples in the Shanghai clinical test according to the present invention.
FIG. 27 shows 30 RNA internal controls (ABI 7500PCR apparatus) of positive samples.
FIG. 28 is a map of the channels of 30 positive samples 2019-nCoV (ABI 7500PCR instrument).
FIG. 29 is a negative sample 20 RNA internal reference pictures, 3 sample RNA internal reference Cts have no jump and can be indicated as sampling invalid samples (ABI 7500PCR instrument).
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 primer and Probe design and screening
1. The invention analyzes several virus sequences and designs primer and probe groups shown in tables 1-4 through a large amount of research and exploration:
TABLE 1 primer and Probe combinations of the invention
Figure 563290DEST_PATH_IMAGE001
TABLE 2 control group 1
Figure 903267DEST_PATH_IMAGE002
TABLE 3 control 2
Figure 479741DEST_PATH_IMAGE003
TABLE 4 control 3
Figure 547055DEST_PATH_IMAGE004
2. Meanwhile, through the optimization exploration of a suitable detection system, the following detection system is established: 23.5. mu. lRT-PCR amplification solution and 1.5. mu.l of enzyme mixture were added to each PCR tube. And a 5. mu.l amount of template RNA was added.
Namely, the RT-PCR detection system (total volume 30. mu.l) comprises:
(1) 23.5 mul RT-PCR amplification solution, wherein the amplification solution comprises a primer, a probe and a PCR buffer solution;
the using concentration of the probe for detecting the target gene is 200 nM, the using concentration of the probe for detecting the internal standard is 100nM, the using concentrations of the upstream primer and the downstream primer for detecting the target gene are 200 nM, and the using concentration of the primer for detecting the internal standard is 100 nM;
the composition of the PCR buffer was: 28mol/L Tris-HCl pH8.0, 20mmol/L (NH)4)2SO4，30mmol/L KC1，4.0mmol/ L MgCl2, 0.2mol/ L dNTPs；
(2) 1.5 mul of enzyme mixture (including hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG));
(3) mu.l of template nucleic acid.
3. The primers and probes in the groups shown in the tables 1-4 are used for detection, and the new crown 2019-nCoV quality control product, the influenza A (Flu A) quality control product, the influenza B (Flu A) quality control product, the respiratory syncytial virus A and the internal standard quality control product are used for carrying out 4 groups of primer and probe combination for testing.
The fluorescence detection channel was set as shown in Table 5
TABLE 5
Figure 17219DEST_PATH_IMAGE005
The RT-PCR amplification program was set up as shown in Table 6:
TABLE 6
Figure 636419DEST_PATH_IMAGE006
4. The results are shown in Table 7 below.
TABLE 7 Ct values of MIX detection results of each group
Figure 575556DEST_PATH_IMAGE007
The results show that the primer-probe combinations of the present invention are optimal in detection sensitivity and specificity for each pathogen, as compared to the control groups 1 to 3. One of the greatest advantages of the primer probe combination is that 6 pairs of primers and 6 probes are in total in the same PCR reaction system, namely, the synchronous detection of multiple pairs of primers and probes for different pathogens is realized, the sensitivity and specificity of detection for each pathogen can meet the clinical detection requirements, and the design of the primer probes is of great importance.
EXAMPLE 2 kit
1. The novel nucleic acid joint detection kit for the coronavirus, the influenza A virus, the influenza B virus and the respiratory syncytial virus comprises the following components:
(1) primer and probe combination
The present inventors obtained, through extensive exploratory optimization by analyzing DNA of clinical specimens, SEQ ID NOs: 1-2 and SEQ ID NO: 3 with a probe; SEQ ID NO: 4-5 and SEQ ID NO: 6; SEQ ID NO: 7-8 and SEQ ID NO: 9 with a probe; SEQ ID NO: 10-11 and SEQ ID NO: 12 with a probe; SEQ ID NO: 13-14 and SEQ ID NO: 15 with a probe; SEQ ID NO of the nucleotide sequence of the internal standard: 16-17 and SEQ ID NO: 18 in the above list. A first set of probes labeled with a maximum emission wavelength of 518nm dye, a second set of probes labeled with a maximum emission wavelength of 553nm dye, a third set of probes labeled with a maximum emission wavelength of 607nm dye, and a fourth set of probes labeled with a maximum emission wavelength of 667nm dye. Therefore, the new coronavirus 2019-nCoV, influenza A (FluA), influenza B (FluB) and respiratory syncytial virus are detected, and the intracellular B2M gene is simultaneously detected for evaluating the sample quality and the PCR inhibitor.
Preferably, the concentration of the probe used for detecting the target gene is 180-220nM, the concentration of the probe used for detecting the internal standard is 80-120 nM, the concentration of the upstream and downstream primers used for detecting the target gene is 180-220nM, and the concentration of the primer used for detecting the internal standard is 80-120 nM.
More preferably, the target gene is detected using a probe at a concentration of 200 nM, the internal standard is detected using a probe at a concentration of 100nM, the upstream and downstream primers for the target gene are detected using a concentration of 200 nM, and the internal standard is detected using a concentration of 100 nM.
(2) PCR buffer solution:
the PCR buffer solution comprises the following components: 26-30mol/L pH8-8.5 Tris-HCl, 15-25mmol/L (NH)4)2SO4，28-32mmol/L KC1，3.5-4.5mmol/ L MgCl2，0.15-0.25mol/ L dNTPs。
Preferably, the PCR buffer consists of: 28mol/L Tris-HCl pH8.0, 20mmol/L (NH)4)2SO4，30mmol/L KC1，4.0mmol/ L MgCl2, 0.2mol/ L dNTPs。
(3) Enzyme mixture liquid:
the enzyme mixed solution comprises hot start Taq enzyme, reverse transcriptase and uracil glycosylase (UNG); wherein the dosage of the hot start Taq enzyme is 3-8U/person, the dosage of the reverse transcriptase is 3-8U/person, and the dosage of the uracil glycosylase is 0.05-0.2U/person.
(4) Blank control
The blank control is rnase-free water;
(5) positive control:
the positive control: 2019-nCoV ORF1ab gene plasmid with concentration of 1.0X 105copies/ml; 2019-nCoVN gene plasmid with the concentration of 1.0 multiplied by 105copies/ml; influenza A virus plasmid at a concentration of 1.0X 105copies/ml; influenza B virus plasmid at a concentration of 1.0X 105copies/ml; respiratory syncytial virus plasmid at a concentration of 1.0X 105copies/ml; internal standard plasmid at a concentration of 1.0X 104copies/ml。
2. The use method of the kit comprises the following steps:
(1) sample collection and processing:
1) nasopharyngeal swab: the secretion from nasal cavity and pharynx is wiped with a sterile swab, and then placed in a sterile test tube (400-.
2) Sputum: rinsing with clear water for 3 times before rinsing, and forcibly expectorating sputum in deep respiratory tract to a sterile sputum collector. When the patient has deep sputum and is not easy to expectorate, the back can be beaten before expectoration to assist in expectoration. The amount of collected sputum cannot be less than 1 mL. The liquefaction method comprises the following steps: adding acetylcysteine (10 g/L) with the same volume into the sputum sample, oscillating for 30 minutes at room temperature, and carrying out subsequent nucleic acid extraction after the sputum sample is fully liquefied.
3) Alveolar lavage fluid: collecting bronchoalveolar lavage fluid, sealing and inspecting.
The sample adopts a MagaBio plus virus DNA/RNA purification kit II matched with an automatic extraction instrument of Bori.
(2) Sample detection:
adding 5 mul of DNA extracted from the sample into the PCR reaction solution, uniformly mixing, placing in a fluorescent quantitative PCR instrument for reaction, and carrying out an amplification procedure: 55 ℃ for 15 minutes, hot start at 95 ℃ for 30 seconds, thermal cycling at 95 ℃ for 10 seconds, 60 ℃ for 35 seconds, for a total of 45 cycles.
(3) Post amplification curve analysis:
1) the Ct value of the sample in FAM, HEX/JOE and ROX fluorescence detection channels is shown as Undet, the Ct value of the RNA internal reference B2M in CY5 fluorescence detection channel is less than or equal to 40, and the sample is judged to be negative.
2) The Ct value of the sample in the FAM fluorescence detection channel is less than or equal to 40, the Ct values of the HEX/JOE and ROX fluorescence detection channels are shown as Undet, and the Ct value of the RNA internal reference B2M in the CY5 fluorescence detection channel is less than or equal to 40 or Undet, and the sample is judged to be positive for the influenza A virus/the influenza B virus.
3) The Ct value of the sample in the HEX/JOE fluorescence detection channel is less than or equal to 40, the Ct values of the FAM and ROX fluorescence detection channels are shown as Undet, and the Ct value of the RNA internal reference B2M in the CY5 fluorescence detection channel is less than or equal to 40 or Undet, and the sample is judged to be positive by the respiratory syncytial virus.
4) The Ct value of the sample in the ROX fluorescence detection channel is less than or equal to 40, the Ct values of the FAM and HEX/JOE fluorescence detection channels are shown as Undet, and the Ct value of the RNA internal reference B2M in the CY5 fluorescence detection channel is less than or equal to 40 or Undet, and the sample is judged to be 2019-nCoV positive.
5) And (4) displaying the Ct value of the sample in each fluorescence channel as Undet, judging that the sample is invalid, and resampling.
Example 3 test example
1. Experiment of
Aiming at the detection accuracy and effectiveness of the new coronavirus 2019-nCoV, influenza A, influenza B and respiratory syncytial virus, the kit provided by the invention is used for detecting the detection accuracy and effectiveness of the new coronavirus 2019-nCoV, the influenza A, the influenza B and the respiratory syncytial virus, the new coronavirus plasmid quality control product and the internal standard plasmid quality control product (different concentrations are 1 multiplied by 10)6copies/ml、1×105copies/ml、1×104copies/ml、1×103copies/ml、1×102copies/ml、1×101copies/ml), and Guangdong CDC New coronal ventricular quality assessment control 5330 (dilution of different concentrations: 1X 105copies/ml、1×104copies/ml、1×103copies/ml、1×101copies/ml), 18 cases of quality control products of the nation and moral corporation.
2. Results
The results are shown in fig. 1-6, which are graphs of the amplification results of the first fluid quality control product, the second fluid quality control product, the respiratory syncytial virus quality control product, the new corona quality control product, the internal standard quality control product and the new corona ventricular mass evaluation quality control product 5330 with different concentrations, and the results of the CT values of the amplification results are shown in table 8. The result shows that the minimum detection limit of the kit aiming at the new coronavirus 2019-nCoV, influenza A, influenza B and respiratory syncytial virus is 1 multiplied by 103copies/ml。
TABLE 8
Figure 197293DEST_PATH_IMAGE008
Meanwhile, the results of the detection of 18 cases of quality control products of the Pondersheng company comprise human coronavirus (HCoV-OC 43, HCoV-229E, HCoV- -HKU1, HCoV- -NL 63), influenza A (H1N 1, H5N1, H7N9, H9N2, INFA), respiratory syncytial virus A, influenza B, dengue virus I, human parainfluenza 2 (PIV 2 RNA), human parainfluenza 3 (PIV 3 RNA), mycoplasma pneumoniae, adenovirus and hepatitis B, the amplification curves are respectively shown in FIGS. 7-24, and the results show that the virus detection is non-crossed, and the detection results of the new coronavirus, the influenza A, the influenza B and the respiratory syncytial virus are in accordance with expectations.
The amplification results of the positive control and the blank control in the kit are shown in FIG. 25.
Example 4 test example
In order to detect the accuracy and effectiveness of the detection of the novel coronavirus 2019-nCoV, influenza A, influenza B and respiratory syncytial virus by the kit, the nucleic acid detection capability verification intercellular quality control product of the novel coronavirus (2019-nCoV) issued by Shanghai clinical detection center is verified, the result is 100% coincidence, an amplification curve chart is shown in FIG. 26, and the specific result is shown in Table 9:
table 9: new crown 2019-nCoV nucleic acid interstitial disease detection result of Shanghai clinical detection center
Figure 397330DEST_PATH_IMAGE009
Example 5 test example
Detection verification result of Guangdong province disease prevention control center
Sample type: nucleic acid extract
Number of samples: 50 cases, 30 cases positive, 20 cases negative. The positive sample is extracted by adopting a purelink ViralRNA/DNA Mini kit of the Weijie Weiji corporation. The negative sample was extracted using an automatic extractor from Thermoscientific corporation, PrefiledViral Total NA kit.
A fluorescent PCR instrument: ABI7500 fluorescent PCR instrument.
The results of the clinical specimen tests are shown in tables 10 and 11. The fluorescent PCR amplification curves are shown in FIGS. 27-29.
After 30 positive samples are confirmed by sequencing, the detection result is consistent with the sequencing result.
And (4) conclusion: the coincidence rate of 30 positive samples is 100%, the original laboratory detection result of 3 negative samples in 20 negative samples is negative, the detection result of the kit monitors no jump of the internal reference, the sample is judged to be an invalid sample, and the detection results of the other samples are coincident.
Tests prove that the detection kit has higher consistency on the results provided by the Xinguan detection and the CDC in Guangdong province.
Test results of clinical positive samples in Table 1030
Figure 62798DEST_PATH_IMAGE010
TABLE 1120 detection results of negative samples and control samples
Figure 738499DEST_PATH_IMAGE011
By combining the experimental results, the kit has high consistency with the actual clinical detection results, has quick and accurate effect on the diagnosis of diseases, and completely meets the requirements of clinical application.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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Claims (10)

1. A group of novel nucleic acid joint detection primers and probe combinations for coronavirus, influenza A virus, influenza B virus and respiratory syncytial virus are characterized by comprising the following components:
(1) the nucleotide sequence of the primer pair and the probe for detecting the 2019-nCoV ORF1ab target gene is shown as SEQ ID NO: 1-3;
(2) the primer pair and the probe for detecting the 2019-nCoV nucleocapsid protein N target gene have nucleotide sequences shown as SEQID NO: 4-6;
(3) the nucleotide sequences of the primer pair and the probe for detecting the target gene of the influenza A virus are shown as SEQ ID NO: 7-9;
(4) the nucleotide sequences of the primer pair and the probe for detecting the target gene of the influenza B virus are shown as SEQ ID NO: 10-11 and SEQ ID NO: 12 is shown in the specification;
(5) the nucleotide sequences of the primer pair and the probe for detecting the target gene of the respiratory syncytial virus are shown as SEQ ID NO: 13-15;
(6) the nucleotide sequences of the primer pair and the probe for detecting the internal standard B2M are shown as SEQ ID NO: 16-18.
2. The primer and probe combination of claim 1, wherein the two ends of the probe are labeled with different dyes with one of the maximum emission wavelengths of 518nm, 538nm to 553nm, 574nm to 575nm, 607nm to 615nm, 640nm, 666nm to 667nm and 690 nm.
3. The combination of the primer and the probe as claimed in claim 1, wherein the probe is labeled with ROX, FAM, HEX or CY5 fluorescence generating group at the 5 'end and MGB at the 3' end.
4. The primer and probe combination of claim 1, wherein the primer of SEQ ID NO: 3 and SEQ ID NO: 6, marking a ROX fluorescence generating group at the 5 'end of the probe and marking MGB at the 3' end;
SEQ ID NO: 9 and SEQ ID NO: 12, the 5 'end of the probe is marked with FAM fluorescence generating group, and the 3' end is marked with MGB
SEQ ID NO: 15, marking a HEX fluorescence generating group at the 5 'end of the probe shown in the figure, and marking MGB at the 3' end;
SEQ ID NO: 18 is labeled with CY5 fluorophore at the 5 'end and MGB at the 3' end.
5. Use of the primer and probe combination according to any one of claims 1 to 4 for the preparation of a novel combined nucleic acid detection kit for coronavirus, influenza a virus, influenza b virus and respiratory syncytial virus.
6. A novel nucleic acid combined detection kit for coronavirus, influenza A virus, influenza B virus and respiratory syncytial virus, which is characterized by comprising the primer and the probe combination according to any one of claims 1 to 4.
7. The kit as claimed in claim 6, wherein the concentration of the probe used for detecting the target gene is 180-220nM, the concentration of the probe used for detecting the internal standard is 80-120 nM, the concentration of the upstream and downstream primers used for detecting the target gene is 180-220nM, and the concentration of the primer used for detecting the internal standard is 80-120 nM.
8. The kit of claim 6, further comprising a PCR buffer consisting of: 26 to 30mol/LpH8-8.5 Tris-HCl，15-25mmol/L (NH4)2SO4，28-32mmol/L KC1，3.5-4.5mmol/ L MgCl2，0.15-0.25mol/ L dNTPs。
9. The kit of claim 6, further comprising an enzyme cocktail comprising hot start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG); wherein the dosage of the hot start Taq enzyme is 3-8U/person, the dosage of the reverse transcriptase is 3-8U/person, and the dosage of the uracil glycosylase is 0.05-0.2U/person.
10. The kit of claim 6, further comprising a blank control and a positive control;
the blank control is rnase-free water;
the positive control: 2019-nCoV ORF1ab gene plasmid with concentration of 1.0X 105copies/ml; 2019-nCoV N gene plasmid with the concentration of 1.0 multiplied by 105copies/ml; influenza A virus plasmid at a concentration of 1.0X 105copies/ml; influenza B virus plasmid at a concentration of 1.0X 105copies/ml; respiratory syncytial virus plasmid at a concentration of 1.0X 105copies/ml; internal standard plasmid at a concentration of 1.0X 104copies/ml。
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