CN107529559B - Multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus and application - Google Patents
Multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus and application Download PDFInfo
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Abstract
The invention relates to a multiple fluorescence PCR detection kit for hand-foot-and-mouth disease viruses and application thereof, comprising a primer pair for amplifying common hand-foot-and-mouth disease pathogens and a primer and a probe for quality control internal standard genes, wherein the sequences of the primer and the probe are shown as SEQ ID NO.1-SEQ ID NO. 12; the invention has good specificity to the primers and probes used for enterovirus 71, coxsackievirus A16 and enterovirus universal types, and avoids the problem that different primers/probes are mutually crosslinked; in addition, the kit of the invention is designed with an internal standard, the internal standard template is a housekeeping gene and participates in sample extraction and PCR amplification, the whole reaction process can be effectively monitored, and false negative is prevented; and can effectively avoid the inhibition of certain inhibitors on PCR reaction.
Description
Technical Field
The invention relates to a kit, in particular to a multiple fluorescence PCR detection kit for hand-foot-and-mouth disease viruses and application thereof.
Background
Hand-foot-and-mouth disease (HFMD) is one of common infectious diseases caused by various enteroviruses, has the characteristics of fast dissemination and strong epidemic and can cause large-scale epidemic in a short time. Most children under 5 years of age have the highest incidence in the age group of less than 3 years of age. Infants and children are easy to infect the hand-foot-and-mouth disease in 4-9 months every year, and the total number of sick infants generally reaches a peak in 5-7 months. The propagation modes mainly include: people are in close contact with the virus-polluted toys, towels, milk products, close-fitting clothes, bedding and the like; the virus in the secretion and saliva of throat of a patient can be transmitted through air (spray) or through mouth; drinking or eating water or food contaminated with viruses may also lead to infections. The symptoms of most patients are mild, fever and herpes or rash accompanied with hands, feet, oral cavity and other parts are used as main symptoms, and a few children patients can cause myocarditis, pulmonary edema, aseptic meningitis, meningoencephalitis, acute flaccid paralysis, respiratory tract infection and other complications.
More than 20 enteroviruses capable of causing hand-foot-and-mouth disease belong to the human enterovirus genus of Picornaviridae (Picornaviridae). Types 16, 4, 5, 9 and 10 of Coxsackievirus A group, types 2 and 5 of B group and Enterovirus 71 (Enterovirus 71, EV71) are common pathogens of hand-foot-and-mouth diseases, and the Coxsackievirus A16 (Coxsackievirus A16, CA16) and the Enterovirus 71 (EV71) are the most common pathogens.
The fluorescence PCR detection technology has the advantages of high sensitivity, rapid detection and the like, and is expected to be widely applied to clinical diagnosis. However, the traditional fluorescent real-time PCR can only amplify nucleic acid of one pathogen at a time, multiple attempts are needed to accurately find out the pathogenic pathogen, a lot of time and energy are consumed, infection caused by multiple pathogens cannot be effectively distinguished, and only single-index detection can be performed on EV71, CA16 and other enteroviruses in different reaction tubes respectively, all viruses cannot be detected at one time, so that the detection efficiency is low, and the detection cost is high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a multiple fluorescence PCR detection kit for hand-foot-and-mouth disease viruses and application thereof, wherein the kit can realize that whether a sample to be detected is infected with enterovirus 71 (EV71), coxsackievirus A16 (CA16) and enterovirus universal type (EVU) can be detected rapidly and simultaneously in one reaction system, so that three common enteroviruses in human throat swabs, feces and herpes fluid can be detected rapidly, accurately and effectively.
The technical scheme of the invention is as follows: a multiple fluorescence PCR detection kit for hand-foot-and-mouth disease viruses comprises the following primer pairs for amplifying common hand-foot-and-mouth disease pathogens;
(1) primers and probes for detecting enterovirus type 71 (EV 71):
a forward primer: the DNA sequence is shown in SEQ ID NO. 1;
reverse primer: the DNA sequence is shown in SEQ ID NO. 2;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 3;
(2) and primers and probes for detecting coxsackievirus A16 type (Cox A16):
a forward primer: the DNA sequence is shown in SEQ ID NO. 4;
reverse primer: the DNA sequence is shown in SEQ ID NO. 5;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 6;
(3) primers and probes for detecting enterovirus universal type (EVU):
a forward primer: the DNA sequence is shown in SEQ ID NO. 7;
reverse primer: the DNA sequence is shown in SEQ ID NO. 8;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 9;
and primers and probes for quality control of internal standard genes:
a forward primer: the DNA sequence is shown in SEQ ID NO. 10;
reverse primer: the DNA sequence is shown in SEQ ID NO. 11;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 12;
wherein the primer and the probe are used together in one detection.
Preferably, one end of the probe of the enterovirus 71 (EV71) is marked with a fluorescent group FAM, and the other end is marked with a quenching group BHQ 1;
preferably, one end of the probe of the coxsackievirus A16 type (Cox A16) is marked with a fluorescent group HEX, and the other end is marked with a quenching group BHQ 1;
preferably, one end of the enterovirus universal (EVU) probe is marked with a fluorescent group ROX, and the other end is marked with a quenching group BHQ 2;
preferably, one end of the probe of the internal standard gene is marked with a fluorescent group Cy5, and the other end is marked with a quenching group BHQ 2.
Wherein, the proportion of primers and probes of the enterovirus 71 (EV71) is respectively as follows: SEQ ID NO. 1: SEQ ID NO. 2: SEQ ID NO.3 is 2: 2: 1;
the proportions of the primer and the probe of the coxsackievirus A16 type (Cox A16) are respectively as follows: SEQ ID NO. 4: SEQ ID No. 5: SEQ ID No.6 is 2: 2: 1;
the proportions of primers and probes of the enterovirus universal type (EVU) are respectively as follows: SEQ ID NO. 7: SEQ ID NO. 8: SEQ ID NO.9 is 2: 2: 1;
the proportion of the primer and the probe of the internal standard gene is respectively as follows: SEQ ID NO. 10: SEQ ID NO. 11: SEQ ID No.12 is 2: 2: 1.
the kit also comprises a PCR reaction buffer solution, an enzyme system, a positive control and a negative control;
further, the PCR reaction buffer solution consists of 5 multiplied PCR buffer, dNTPs and DEPC water;
further, the enzyme system comprises hot start Taq enzyme, reverse transcriptase and RNase inhibitor,
further, the positive control is pseudovirus containing enterovirus 71 (EV71), coxsackievirus A16 (CA16) and enterovirus universal type (EVU) target gene segments;
further, the negative control is a cell culture solution.
The reverse transcriptase is M-MLV enzyme, cDNA formed by reverse transcription of the extracted sample RNA template through the reverse transcriptase M-MLV enzyme is used as a template for PCR amplification; the RNA degradation by the RNase is inhibited by the RNase inhibitor, so that the detection accuracy is improved; the hot start Taq enzyme has a DNA chain extension effect of 5 '→ 3' and reacts with a template DNA, a primer and Mg during a reaction process2+The formed complex is combined and amplified through a PCR process;
the application of the hand-foot-and-mouth disease virus multiplex PCR detection kit in detection of hand-foot-and-mouth disease viruses is also within the protection scope of the invention, and particularly in application of conventional pathogen enterovirus 71 (EV71), Coxsackie virus A16 (CA16) and enterovirus universal type (EVU).
The RT-PCR reaction system for detecting the common hand-foot-and-mouth disease virus by using the kit is 25 mu L: wherein, the enzyme system 2 mu L, PCR reaction buffer solution 16 mu L, EV71/CA16/EVU primer and probe mixed solution 2 mu L, template 5 mu L;
RT-PCR reaction conditions:
keeping reverse transcription at 50 ℃ for 25min, and keeping hot start at 95 ℃ for 5 min; maintaining denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 15s, extending at 72 ℃ for 30s, and pre-amplifying for 5 cycles; the denaturation was maintained at 95 ℃ for 10s, annealing/extension was maintained at 60 ℃ for 45s, and the fluorescence signal was collected for 40 cycles.
Selecting a detection channel of the PCR instrument:
a FAM channel is selected as an enterovirus 71 type fluorescence signal, an HEX/VIC channel is selected as a coxsackievirus A16 type fluorescence signal, an ROX channel is selected as an enterovirus general type fluorescence signal, and a Cy5 channel is selected as an internal standard fluorescence signal.
The invention has the beneficial effects that: (1) the invention optimizes the reaction parameters, analyzes the detection limit, the precision and other properties of enterovirus 71, coxsackievirus A16 and enterovirus general type, can realize one-time reaction and simultaneously detect three common pathogens causing intestinal system infection, shortens the detection time, has high accuracy and provides a powerful detection tool for clinic and detection laboratories;
(2) the invention has good specificity to the primers and probes used for enterovirus 71, coxsackievirus A16 and enterovirus universal types, and avoids the problem that different primers/probes are mutually crosslinked, and each group of primers and probes can not generate cross homology to other target and non-target nucleic acid sequences;
(3) the kit of the invention designs an internal standard, the internal standard template is a housekeeping gene, and participates in sample extraction and PCR amplification, so that the whole reaction process can be effectively monitored, and false negative is prevented;
(4) can effectively avoid the inhibition of certain inhibitors on PCR reaction: a clinical sample of the enterovirus generally contains more host interfering substances, a plurality of the substances inhibit PCR enzyme, and a multiplex reverse transcription real-time fluorescence quantitative PCR amplification system can effectively avoid the inhibition of the inhibitors on PCR reaction.
Drawings
FIG. 1a is the EV71 amplification curve of the positive reference P1-P10 in example 3;
FIG. 1b is the CA16 amplification plot of the positive reference P1-P10 of example 3;
FIG. 1c is the EVU amplification plot of the positive references P1-P10 of example 3;
FIG. 2 is a graph showing the amplification curves of the negative reference products N1-N10 in example 3;
FIG. 3a is a graph of EV71 amplification of the detection limit reference L1 in example 3;
FIG. 3b is a graph of the EVU amplification of detection limit reference L1 of example 3;
FIG. 3c is a graph of the amplification of CA16 of the detection limit reference L2 in example 3;
FIG. 3d is a graph of the EVU amplification of detection limit reference L2 of example 3;
FIG. 4a is the amplification curve of the references R1-R4 in example 3 with precision;
FIG. 4b is the EV71 amplification curve of the precision reference R1-R4 in example 3;
FIG. 4c is the CA16 amplification curve of the precision references R1-R4 of example 3;
FIG. 4d is the EVU amplification profile of references R1-R4 with precision in example 3;
FIG. 5 is a graph showing the amplification curve of the interference substance versus the result of PCR detection in example 3.
Detailed Description
The following further describes embodiments of the present invention with reference to the accompanying drawings:
example 1 PCR detection kit
The enterovirus 71 type (EV71), the coxsackievirus A16 type (Cox A16), the enterovirus universal type (EVU), the primers of the internal standard genes and the probes are shown in a table 1, wherein all the primers in the table 1 are synthesized by the company of Biotechnology engineering (Shanghai);
TABLE 1 primers and probes for Enterovirus 71 (EV71), Coxsackie virus A16 (CoxA16), Universal Taoise Virus (EVU), internal reference genes
The kit consists of PCR reaction buffer solution, an enzyme system, a positive control, a negative control, all primer pairs and probes in the table 1;
table 2 shows the components of EV71/CA16/EVU primer probe mixture (batch: 1000 parts)
| Serial number | Name of raw materials | Specification of | Dosage of | |
| 1 | EV71F | 100μmol/ | 50μl | |
| 2 | EV71R | 100μmol/L | 50μl | |
| 3 | EV71P | 100mmol/ | 25μl | |
| 4 | CA16F | 100μmol/L | 80μl | |
| 5 | CA16R | 100μmol/ | 80μl | |
| 6 | CA16P | 100μmol/L | 40μl | |
| 7 | EVUF | 100μmol/ | 60μl | |
| 8 | EVUR | 100μmol/L | 60μl | |
| 9 | EVUP | 100μmol/ | 20μl | |
| 10 | IC.F | 100μmol/L | 30μl | |
| 11 | IC.R | 100μmol/ | 30μl | |
| 12 | IC.P | 100μmol/L | 15μl | |
| 13 | DEPC water | —— | 1.56ml | |
| Total volume | —— | 2ml |
Table 3 shows the components of the enzyme system (batch: 1000 parts by weight)
| Serial number | Name of raw materials | Specification of | Dosage of |
| 1 | Hot start Taq enzyme | 5U/μl | 500μl |
| 2 | Hot start Taq enzyme activator | 100× | 250μl |
| 3 | M-MLV reverse transcriptase | 200U/μl | 300μl |
| 4 | RNase inhibitors | 40U/μl | 200μl |
| 5 | M-MLV reverse transcriptase diluent | —— | 750μl |
| Total volume | —— | 2.0ml |
Table 4 shows the PCR reaction buffer composition (batch: 1000 copies)
| Serial number | Raw materialsName (R) | Specification of | Dosage of |
| 1 | 5×PCR buffer | 5× | |
| 2 | dNTPs | 2.5mmol/L | 2ml |
| 3 | DEPC water | —— | 9.0ml |
| Total volume | —— | 16.0ml |
EV71/CA16/EVU positive control stock solution (1.0X 10)9PFU/ml), diluted 10 with physiological saline4Doubling, and adding BSA with the final concentration of 1% to prepare EV71/CA16/EVU positive control;
taking A549 cell culture solution (concentration is 1 × 10)6Individual cells/ml) was dispensed, which was the negative control.
Example 2 methods for detecting Enterovirus 71 (EV71), Coxsackie virus A16 (CoxA16), and Enterovirus Universal type (EVU)
(1) Extraction of viral nucleic acids
Extracting nucleic acid from the sample using QIAamp MinElute Virus Spin Kit (cat # 57704) from QIAGEN or viral genomic DNA/RNA extraction Kit (cat # DP315) from Tiangen Biochemical technology Ltd;
(2) arrangement of reaction System
Taking 16 mu L Xn of PCR reaction buffer solution, 2 mu L Xn of enzyme system, 2 mu L Xn of EV71/CA16/EVU primer and 2 mu L Xn of probe mixed solution according to the number n of reaction tubes, fully dissolving and uniformly mixing the PCR reaction buffer solution, the EV71/CA16/EVU primer and the probe mixed solution before use, centrifuging the enzyme system to ensure that all enzymes are concentrated at the bottom before use,
subpackaging the PCR reaction solution into PCR reaction tubes according to 20 mu L/tube, and moving the reaction tubes filled with the PCR reaction solution to a sample processing area;
(3) sample application
Respectively sucking the processed sample, the negative control and the positive control by a suction nozzle with a filter element by 5 microlitres, then respectively adding the samples, the negative control and the positive control into a PCR reaction tube filled with PCR reaction liquid, covering a tube cover, centrifuging for several seconds after marking, and then moving to an amplification detection area;
(4) fluorescent quantitative PCR amplification
Putting a PCR reaction tube in an ABI7500 or SLAN96P fluorescent PCR instrument for amplification detection, and simultaneously detecting enterovirus 71 type (EV71), Coxsackie virus A16 type (CoxA16) and enterovirus universal type (EVU) (the fluorescent PCR instrument contains FAM, HEX/VIC, ROX and Cy5 fluorescent channels);
② fluorescent RT-PCR reaction conditions:
keeping reverse transcription at 50 ℃ for 25min, and keeping hot start at 95 ℃ for 5 min; maintaining denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 15s, extending at 72 ℃ for 30s, and pre-amplifying for 5 cycles; maintaining denaturation at 95 ℃ for 10s, annealing/extending at 60 ℃ for 45s, collecting fluorescence signals, and performing 40 cycles;
selecting a detection channel of the PCR instrument: an FAM channel is selected for enterovirus 71 type fluorescent signals, an HEX/VIC channel is selected for coxsackievirus A16 type fluorescent signals, an ROX channel is selected for enterovirus general type fluorescent signals, and a Cy5 channel is selected for internal standard fluorescent signals (note: ABI7500 quenching group and fluorescent correction signal selecting none);
(5) quality control standard
Negative control: the result is negative, the internal standard result is positive and the Ct value is less than 30;
positive control: the result is positive, and the Ct value of the positive control is not more than or equal to the pair;
and thirdly, the two items need to be met simultaneously in one experiment, otherwise, the experiment is invalid, and the experiment should be carried out again.
(6) Analysis of the results
And (4) judging a negative result: if the sample is in the FAM channel, the HEX/VIC channel and ROX channel amplification curves are not S-shaped, the Ct value is UNDET or is more than or equal to 35.00, the internal standard Cy5 channel amplification curve is S-shaped, and the Ct value is less than 30, the result is negative; if the internal standard Cy5 channel amplification curve is not S-shaped or the Ct value of the result is more than or equal to 30, the sample is subjected to rechecking;
and (3) judging a positive result: if the sample is S-shaped in the FAM channel amplification curve and the Ct value is less than or equal to 33, the result is positive EV71, if the sample is S-shaped in the HEX/VIC channel amplification curve and the Ct value is less than or equal to 33, the result is positive CA16, and if the sample is S-shaped in the ROX channel amplification curve and the Ct value is less than or equal to 33, the result is positive EVU;
experimental gray scale region: if the sample is in S shape in FAM channel, HEX/VIC channel or ROX channel amplification curve and the Ct value is 33< Ct <35, the result is located in the experimental gray area, and the sample is subjected to retest.
The kit is used for detecting samples of 593 clinical suspected patients with hand-foot-and-mouth diseases, and the detection results are compared with a patent (201110079878.5), wherein the comparison result of the enterovirus 71 type is shown in Table 5, the Coxsackie virus A16 type is shown in Table 6, the universal type of the enterovirus is shown in Table 7,
TABLE 5 Enterovirus 71 type test results
TABLE 6 detection results of Coxsackie virus type A16
TABLE 7 Universal detection results for enteroviruses
Example 3 kit Performance analysis
1. Positive compliance rate: taking a positive reference product P1-P10 as a sample to be detected, detecting the positive coincidence rate of the positive reference sample by using an ABI7500 real-time fluorescence quantitative PCR instrument detection kit, wherein the sample to be detected P1-P10 are shown in Table 8, and the detection result shows that P1-P10 are all positive, the coincidence rate is 100% (10/10), wherein P1, P2, P7, P8, P9 and P10 are positive for EV71, as shown in figure 1 a; p3, P4, P7, P8, P9, P10 are CA16 negative, as shown in fig. 1 b; P1-P9 were all EVU positive, as shown in FIG. 1 c;
TABLE 8 Positive reference sample to be tested
2. Negative coincidence rate: taking negative reference products N1-N10 as samples to be detected, detecting on an ABI7500 real-time fluorescence quantitative PCR instrument as shown in Table 9, analyzing the coincidence rate of the negative reference products of the kit, and displaying the result that the EV71/CA16/EVU detection results of the negative reference products N1-N10 are all negative, wherein the coincidence rate is 100% (10/10), and the sample is specifically shown in figure 2;
TABLE 9 sample of negative reference to be tested
| Numbering | Sample(s) | Working concentration |
| N1 | A549 cell culture solution | 1.0×106/ml |
| N2 | Influenza A H1N1 virus | 1.0×105PFU/ml |
| N3 | Influenza B virus | 1.0×105PFU/ml |
| N4 | Human respiratory syncytial virus type A | 1.0×105PFU/ml |
| N5 | Human adenovirus type 3 | 1.0×105PFU/ml |
| N6 | Human enterovirus 71 type | 1.0×105PFU/ml |
| N7 | Human parainfluenza virus type 1 | 1.0×105PFU/ml |
| N8 | Mumps virus | 1.0×105PFU/ml |
| N9 | Haemophilus influenzae | 1.0×106CFU/ml |
| N10 | Streptococcus pneumoniae | 1.0×106CFU/ml |
3. Detection limit: the inactivated enterovirus 71 and coxsackievirus A16 are respectively diluted to 1 × 103Preparing detection limit reference substances L1 and L2 from PFU/ml, using the kit of the invention to repeatedly detect for 20 times by using the sample to be detected, wherein the detection result of L1 for at least 17 times is positive EV71/EVU, as shown in FIGS. 3a and 3 b; at least 17 times of the L2 test results are positive for CA16/EVU, as shown in FIGS. 3c and 3 d; indicating that the detection limit is not higher than 1 x 103PFU/ml。
4. Precision: diluting Enterovirus 71 to concentration of 2 × 106PFU/ml and 2X 103PFU/ml as precision references R1, R2;
the Coxsackie virus A16 was diluted to a concentration of 2X 106PFU/ml and 2X 103PFU/ml as precision references R3, R4;
the detection is repeated for 10 times by using the kit, and the detection results are shown in FIGS. 4a, 4b, 4c and 4d, wherein the variation Coefficients (CV) of the detection results EV71 and EVU Ct of R1 and R2 are less than or equal to 5%;
the Coefficient of Variation (CV) of the CA16 and EVU Ct values of the detection results of R3 and R4 is less than or equal to 5 percent.
5. Detection of interfering substances: adding 1 part of normal oropharyngeal swab sample into normal saline, uniformly mixing by shaking, uniformly dividing the sample into 3 parts, wherein one part is added with human blood (the concentration of hemoglobin is more than 60g/L), one part is added with nasopharyngeal secretion (negative pressure suction matter, the concentration of total protein is more than 120g/L), the other remaining part is used as a control, and simultaneously, fecal samples with different characterization differences are selected and comprise water sample feces, bloody feces and hard feces, the normal feces are used as a control, and the samples are all negative to enteroviruses through detection;
to the above 7 samples were added the enterovirus 71, coxsackievirus A16 and enterovirus universal culture mixtures to final concentrations of 1.0X 103PFU/ml, detecting on ABI7500 real-time fluorescence quantitative PCR instrument, verifying that the interfering substance in the sample has interfering effect on the PCR reaction process of the kit, analyzing the no influence of the substance possibly existing in the sample on the detection result by detection, as shown in FIG. 5;
in conclusion, the kit can detect and distinguish enterovirus 71, coxsackievirus A16 and enterovirus general types, and specific experiments prove that other viruses which are the same as infected parts or similar to infected symptoms do not have cross reaction, feces, throat swabs and other substances possibly existing in a sample to be detected do not interfere with the detection result of the kit, and the detection limit is not higher than 1.0 multiplied by 103PFU/ml; the coincidence rate of the positive reference substance and the negative reference substance is 100 percent; the detection limit reference substances L1 and L2 are repeatedly detected for 20 times, the detection result of at least 17 times of L1 is positive by EV71/EVU, and the detection result of at least 17 times of L2 is positive by CA 16/EVU; the precision reference products R1-R4 are repeatedly detected for 10 times, and the variation Coefficient (CV) of the EV71 and EVU Ct values of the detection results of R1 and R2 is less than or equal to 5 percent; the Coefficient of Variation (CV) of the CA16 and EVU Ct values of the detection results of R3 and R4 is less than or equal to 5 percent.
The foregoing embodiments and description have been presented only to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as hereinafter claimed.
Claims (7)
1. A multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus is characterized in that: comprises the following steps for amplification
Primer pairs and probes of common hand-foot-and-mouth disease pathogens:
(1) primers and probes for detecting enterovirus type 71 (EV 71):
a forward primer: the DNA sequence is shown in SEQ ID NO. 1;
reverse primer: the DNA sequence is shown in SEQ ID NO. 2;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 3;
(2) and primers and probes for detecting coxsackievirus A16 type (Cox A16):
a forward primer: the DNA sequence is shown in SEQ ID NO. 4;
reverse primer: the DNA sequence is shown in SEQ ID NO. 5;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 6;
(3) primers and probes for detecting enterovirus universal type (EVU):
a forward primer: the DNA sequence is shown in SEQ ID NO. 7;
reverse primer: the DNA sequence is shown in SEQ ID NO. 8;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 9;
and primers and probes for quality control of internal standard genes:
a forward primer: the DNA sequence is shown in SEQ ID NO. 10;
reverse primer: the DNA sequence is shown in SEQ ID NO. 11;
and (3) probe: the DNA sequence is shown in SEQ ID NO. 12;
wherein the primer and the probe are used together in one detection.
2. The multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus according to claim 1, characterized in that:
one end of the probe of the enterovirus 71 (EV71) is marked with a fluorescent group FAM, and the other end is marked with a quenching group BHQ 1.
3. The multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus according to claim 1, characterized in that:
one end of the probe of the coxsackievirus A16 type (Cox A16) is marked with a fluorescent group HEX, and the other end is marked with a quenching group BHQ 1.
4. The multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus according to claim 1, characterized in that:
one end of the enterovirus universal (EVU) probe is marked with a fluorescent group ROX, and the other end is marked with a quenching group BHQ 2.
5. The multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus according to claim 1, characterized in that:
one end of the probe of the internal standard gene is marked with a fluorescent group Cy5, and the other end is marked with a quenching group BHQ 2.
6. The multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus according to claim 1, characterized in that:
the proportion of primers and probes of enterovirus 71 (EV71) is respectively as follows: SEQ ID NO. 1: SEQ ID NO. 2: SEQ ID No.3 is 2: 2: 1;
the proportions of the primer and the probe of the coxsackievirus A16 type (Cox A16) are respectively as follows: SEQ ID NO. 4: SEQ ID No. 5:
SEQ ID No.6 is 2: 2: 1;
the proportions of primers and probes of the enterovirus universal type (EVU) are respectively as follows: SEQ ID NO. 7: SEQ ID NO. 8: SEQ ID NO
ID NO.9 is 2: 2: 1;
the proportion of the primer and the probe of the internal standard gene is respectively as follows: SEQ ID NO. 10: SEQ ID NO. 11: SEQ ID NO.12
Is that 2: 2: 1.
7. the multiple fluorescence PCR detection kit for hand-foot-and-mouth disease virus according to claim 1, characterized in that:
the kit also comprises a PCR reaction buffer solution, an enzyme system, a positive control and a negative control;
the PCR reaction buffer solution consists of 5 multiplied PCR buffer, dNTPs and DEPC water;
the enzyme system comprises hot start Taq enzyme, reverse transcriptase and RNase inhibitor;
the positive control contains enterovirus 71 (EV71), coxsackievirus A16 (Cox A16) and enterovirus
Pseudoviruses of the viral universal (EVU) gene segment of interest;
the negative control is cell culture fluid.
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| CN109609693A (en) * | 2018-12-26 | 2019-04-12 | 山东艾克韦生物技术有限公司 | Method that is a kind of while detecting a variety of enteroviruses |
| CN109504805A (en) * | 2018-12-28 | 2019-03-22 | 深圳市艾伟迪生物科技有限公司 | The nucleic acid compositions and kit and detection method of four kinds of enteroviruses of detection simultaneously |
| CN110923363B (en) * | 2019-12-19 | 2023-06-27 | 武汉中帜生物科技股份有限公司 | Kit for detecting hand-foot-and-mouth disease pathogen nucleic acid and application thereof |
| CN111088404B (en) * | 2020-02-06 | 2024-01-02 | 广州普世利华科技有限公司 | RDA method and kit for rapidly detecting coxsackievirus A16 and enterovirus 71 |
| CN116286803B (en) * | 2023-02-07 | 2023-11-24 | 广州凯普医药科技有限公司 | Detection primer probe combination and gene chip for synchronously detecting 22 hand-foot-mouth viruses |
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