CN116083652A - Primer, fluorescent freeze-dried microchip, kit and method for detecting monkey pox virus - Google Patents
Primer, fluorescent freeze-dried microchip, kit and method for detecting monkey pox virus Download PDFInfo
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Abstract
The invention relates to the technical field of virus molecular biology detection, and particularly provides a primer, a fluorescent freeze-dried microchip, a kit and a method for detecting monkey pox virus. The sequence of the primer provided by the invention is shown in SEQ ID NO. 1-9. A large number of experiments prove that the primer probe, the kit and the detection method have higher accuracy and excellent specificity than the conventional method, the minimum detection limit can reach 1 copies/mu L, the sensitivity is higher, the intra-batch variation coefficient for detecting the monkey pox virus is between 0.29 and 0.92 percent, the inter-batch variation coefficient is between 0.36 and 1.01 percent, and the repeatability is excellent.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a primer, a fluorescent freeze-dried microchip, a kit and a method for detecting monkey pox virus.
Background
In contrast to RNA viruses, the monkey poxvirus is a large DNA virus, which makes the monkey poxvirus more stable and more efficient than RNA viruses in detecting and repairing mutations. Preliminary genome sequence data indicate that the monkey pox clade is related to the simian clade, which is popular in western africa, and has lighter disease and lower mortality than the non-intermediate clade. The monkey pox infected with human is mainly infected by biting an infected animal, directly contacting the blood, body fluid and monkey pox lesions of an intermediate host or the infected animal. The virus can be transmitted through toxic respiratory droplets, and in addition, the monkey pox can be transmitted through direct contact with the body fluids of the infected person or virus-contaminated articles such as clothing and bedding.
Infection of humans with monkey pox can cause regional lymphadenopathy. The incubation period is 7-14 days, the precursor period is 2-5 days, and the symptoms are fever, general discomfort, fatigue, headache, myalgia, back pain, and sometimes throat pain. Thus, early detection of monkey poxviruses is necessary for both disease control and treatment. Currently, there are high throughput sequencing methods, first generation sequencing methods, and fluorescent PCR methods for the nucleic acid detection method of monkey pox. The high-throughput sequencing method has long time and long time consumption due to the need of a warehouse-building process and long on-machine time. The first generation sequencing method needs to be based on the common PCR, and since the birth, the common PCR is a novel molecular biology technology, a large number of target fragments can be obtained through gene amplification in a short time due to high specificity and sensitivity, the defect that the traditional virus detection technology comprises long virus separation and identification test period is overcome, and a sensitive, quick and practical detection method is provided for the early rapid detection of the monkey pox virus, but the common PCR still has the defects of complex operation, long detection time, easiness in pollution and the like.
Disclosure of Invention
The invention aims to provide a fluorescence freeze-dried microchip, a kit and a method for detecting monkey pox virus, which can effectively detect monkey pox, ensure high accuracy, specificity and sensitivity, complete detection in a short time and obtain a result, and can be transported and stored at normal temperature.
In a first aspect, the present invention provides a primer probe combination, the primers being 5 '-TCTTCCGTC AATGTCTACACAG-3' and 5 '-GGATGCTGATACACGGCCT-3',
the probe is as follows: 5 '-TAATAACGGTGACAGGGTTAAC-3'.
In a second aspect, the present invention provides a fluorescent lyophilized microchip for detecting a monkey pox virus, a fluorescent PCR reaction system for detecting a monkey pox virus being immobilized on the fluorescent lyophilized microchip by lyophilization; the fluorescent PCR reaction system contains the primer probe combination.
In the fluorescent freeze-dried microchip provided by the invention, the fluorescent PCR reaction system further comprises: DNA polymerase, dNTP and Mg 2+ Trehalose, tris-Cl and sterilized deionized water; the fluorescent PCR reaction system in each well is 1-1.2 mu L.
The total volume of the fluorescent PCR reaction system was 36. Mu.L, and the volume of each well was 1.2. Mu.L.
The invention provides a fluorescent freeze-dried microchip, wherein the freeze-drying comprises the following steps: placing the microchip provided with the fluorescent PCR reaction system in a temperature of minus 80 ℃ to minus 96 ℃ for freezing for 1 to 1.2 hours, and then performing equipment freeze-drying;
the apparatus lyophilization comprises: the method comprises a pre-freezing stage, a device vacuumizing stage, a freeze drying stage and a resolution drying stage, wherein the resolution drying stage comprises the following steps: raising the temperature to-25 to-20 ℃ and keeping for 1-1.5h, then raising the temperature of the separator to 30-37 ℃ and keeping for 1.5-2h, and finally lowering the temperature of the separator to 20-25 ℃ and keeping for 1-1.5h.
More specifically, placing the microchip with the fluorescent PCR reaction system at-80 ℃ for freezing for 1 hour, and then performing equipment freeze-drying;
the microchip provided with the fluorescent PCR reaction system is firstly frozen at the temperature of minus 80 ℃ for 1h to be pre-frozen, so that the system in the first pre-freezing stage can be kept in a solid state, and the drying time can be shortened in this step.
Device lyophilization refers to lyophilization in a specialized lyophilization apparatus by setting the operating conditions of the apparatus; preferably, the lyophilization apparatus refers to a vacuum freeze dryer.
Preferably, the apparatus lyophilization comprises: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
The temperature of the partition plate refers to the temperature of a tray partition plate in a freeze dryer, and a microchip provided with the fluorescent PCR reaction system needs to be placed on the tray partition plate; the pre-freezing stage has the function of keeping the fluorescent PCR reaction system in a solid state. The main purpose of the step of vacuumizing the equipment and keeping the freeze drying for 1h is a vacuum freeze dryer, and the internal air is required to be pumped out to sublimate the water in the solid, so that the freeze drying effect is achieved. The next two steps, raising the temperature of the separator to-25 ℃ for 1h, and raising the temperature of the separator to 37 ℃ and for 2h, are to maintain the dry sublimation process; finally, the function of 'cooling the separator to 25 ℃ and keeping for 1 h' is to keep the freeze-dried finished product stable at 25 ℃, so that the whole freeze-drying process is basically completed.
The equipment freeze-drying process is a set of freeze-drying process which is created by the invention aiming at the microchip provided with the fluorescent PCR reaction system. Because the eutectic points of the freeze-drying reagents are different, the measurement is needed, and the heating time and the drying time in the drying process are needed to be optimized. The effect of the components within the lyophilized reagent on the eutectic point is all that is required to be explored by adjusting the reagent component concentration. In particular, the enzyme in the reaction system is placed at normal temperature for a long time, the activity of the enzyme is reduced, and a protective agent and a stabilizer component, such as trehalose 5 mu M, are required to be added; the Tris-Cl 6mM and the concentration of the Tris-Cl 6mM need to be verified and adjusted through experiments to ensure that the excellent effects of high sensitivity, high repeatability, high accuracy, high specificity and the like described by the invention can be obtained when the freeze-dried microchip is used for carrying out fluorescence PCR detection. In addition, the re-dissolution effect of the diluent used after the freeze-drying process is also required to be simultaneously considered, and the excellent effects of high sensitivity, high repeatability, high accuracy, high specificity and the like obtained by the detection method carried on the freeze-dried microchip can be achieved by adjusting the freeze-drying process.
In the fluorescent freeze-dried microchip provided by the invention, more than 30 sample adding holes are formed in the fluorescent freeze-dried microchip; the bottom structure, shape and size of the fluorescent freeze-dried microchip are matched with the sample adding plate of the PCR instrument.
In a third aspect, the present invention provides a kit comprising a fluorescent lyophilized microchip as described above; the kit also comprises a diluent and mineral oil, wherein the diluent is dripped into a sample adding hole of the freeze-dried microchip, and then the freeze-dried microchip is subjected to fluorescent PCR amplification; the mineral oil is used to close the loading wells on the lyophilized microchip.
The kit further comprises: diluting the 10 Xdiluent into 2 Xdiluent by using water without nuclease, dripping the 2 Xdiluent into a sample adding hole of the freeze-dried microchip, and then placing the freeze-dried microchip on a fluorescent PCR instrument for fluorescent PCR amplification. The dilution is specifically PCR 10 Xbuffer, and the specific components do not contain Mg 2+ This dilution is commercially available containing 500mM KCl,100mM Tris-HCl,0.1% gelatin. In use, the dilution is diluted to 2 Xbuffer using nuclease-free water, and 0.6. Mu.L of the solution is added to each well of the microchip.
The kit provided by the invention further comprises: positive plasmid of monkey poxvirus was used as positive control; nuclease-free water served as a negative control.
As a specific test mode of the invention, the invention provides a fluorescent PCR detection kit, which comprises a freeze-dried microchip, a tube of mineral oil, a tube of positive control, a tube of negative control, a tube of diluent and a tube of nuclease-free water.
The freeze-dried microchip comprises a primer, a probe, taq enzyme, trehalose and Tris-Cl, dNTP, mg 2+ . The final concentration of each component of the freeze-drying system of the fluorescent PCR on the freeze-drying microchip is as follows: upstream primer 0.8. Mu.M; 0.8. Mu.M of downstream primer; taqman probe 0.4. Mu.M; DNA polymerase 0.5U/. Mu.L; dNTP 0.4mM; mg of 2+ 4mM; trehalose 5 μm; tris-Cl 6mM; the balance of sterilized deionized water;
the number of the loading holes of the AriaYSB freeze-dried microchip for fluorescence quantitative PCR is 16, 20, 24, 30 or 48, and 1.2 mu L of each microchip hole is added into the fluorescence PCR reaction system (comprising 1 primer probe set of monkey pox virus).
The lyophilized microchip in the kit is coated with primers and T aqman probes having the following nucleotide sequences:
MPV-F：5＇-TCTTCCGTCAATGTCTACACAG-3＇，
MPV-R：5＇-GGATGCTGATACACGGCCT-3＇，
MPV-P：5＇-TAATAACGGTGACAGGGTTAAC-3＇。
as a further refinement, in a specific embodiment, the 3 'end of the probe is labeled with an MGB quencher and the 5' end is labeled with a FAM fluorescent reporter. The "MGB" and "FA M" groups are all fluorescent groups commonly known in the art, and other fluorescent quenching groups and fluorescent reporter groups commonly known in the art may be selected by those skilled in the art to replace the "MGB" and "FAM" herein, for example, common fluorescent quenching groups further include: BHQ-1, BHQ-2, dabcyl 2; common fluorescent reporter groups may also be selected from: TET, HEX, 5-TAMR A, texas Red-X, cy3 (TYE (TM) 563), JOE.
In a further embodiment, the kit further comprises an AriaYSB microchip and conventional reagents for performing fluorescent PCR detection.
The present invention provides the use of the above primer probe combination or the above fluorescent lyophilized microchip or the above kit for detecting monkey pox virus, as understood by those skilled in the art.
In a fourth aspect, the present invention provides a fluorescent PCR detection method for identifying a monkey poxvirus, using the above kit, to perform fluorescent PCR detection on a sample to be detected. The invention provides a fluorescence PCR detection method for identifying monkey pox virus, which belongs to the non-disease treatment and diagnosis purpose.
In some embodiments, the detection is a fluorescent quantitative PCR detection; the lyophilized microchip of the monkey poxvirus comprises: upstream primer 0.8. Mu.M; 0.8. Mu.M of downstream primer; taqman probe 0.4. Mu.MThe method comprises the steps of carrying out a first treatment on the surface of the DNA polymerase 0.5U/. Mu.L; dNTP 0.4mM; mg of 2+ 4mM; trehalose 5 μm; tris-Cl 6mM; the balance was sterile deionized water, in a total volume of 36 μl, 1.2 μl per well volume.
In the fluorescence PCR detection method provided by the invention, the reaction program of fluorescence quantitative PCR comprises the following steps: the temperature is between 40 and 45 ℃ for 5 to 6 minutes, and the cycle is the first step; the temperature is between 90 and 95 ℃ for 1 to 2 minutes, and the second step of circulation is adopted; and (3) detecting fluorescent signals after the extension is finished in the third step of 38-40 cycles at the temperature of 90-95 ℃ for 5-6 s and at the temperature of 55-60 ℃ for 15-20 s.
More specifically, the reaction conditions for the fluorescent PCR of the lyophilized microchip include: 45 ℃ for 5min, which is the first circulation; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
The invention has the beneficial effects that:
according to the invention, a fluorescent PCR method for detecting monkey pox with high speed, accuracy and sensitivity is combined with a chip method, a PCR reaction system is freeze-dried on a microchip, and the temperature rise and fall speed of the microchip is high in the reaction process under the condition that the detection effect is not reduced, so that the detection time is shortened to 30 minutes; the addition of microchip technology enables the whole kit to be stored and transported at normal temperature; the method has the advantages of simple operation, no need of preparing a PCR system, direct addition of diluent, mineral oil and a sample, great simplification of operation, reduction of pollution risk, reduction of reagent consumption and shortening of reaction time, and can provide an advanced and effective early rapid detection technology and monitoring means for the prevention and treatment of the monkey pox, and meanwhile, strive for valuable rapid reaction time for the prevention and treatment of the monkey pox.
(1) According to the invention, specific primers and Ta qman-MGB probes are designed and synthesized according to the published monkey pox viruses, and the monkey pox viruses can be rapidly and sensitively detected by adopting a fluorescent quantitative PCR method.
(2) The invention adopts the high copy target gene on one hand and adopts the Taqma n-MGB probe fluorescence PCR detection method on the other hand, so that the sensitivity is about 100 times of that of the common PCR.
(3) The detection method of the monkey pox virus provided by the invention has the advantages of single tube closed operation pollution prevention, high automation degree, strong specificity, real-time monitoring and the like, and effectively solves the limitation that the traditional PCR method can only detect the end point due to the adoption of a fluorescence detection technology, namely Taqman-MGB fluorescence PCR (Real-time PCR).
(4) Compared with the currently used fluorescent PCR detection technology, the freeze-dried microchip technology has smaller reaction system of only 1.2 mu L and the reaction system of the conventional commercial product of 20-25 mu L, so that the microchip technology can save the consumption of PCR amplification reagents and samples.
(5) The smaller reaction system of the freeze-dried microchip can ensure that the system is heated more uniformly and the temperature rising and falling speed is faster in the PCR amplification process. The temperature rising rate (10-12 ℃/S) is faster than the temperature rising rate (3-5 ℃/S) of the currently adopted fluorescent PCR detection system. The whole fluorescent PCR process (including sample addition) can be completed within 30 minutes, and a computer automatically reports the result without electrophoresis and other subsequent work. After the amplification product carrier chip is used, special treatment is not needed, so that laboratory pollution and false positive detection caused by generated product aerosol are avoided. The operation is convenient and the pollution is reduced.
(6) The freeze-dried microchip can be stored and transported at normal temperature, so that repeated freeze thawing of the reagent is avoided, and the detection result is more stable.
The invention adopts a Real-time fluorescence PCR technology (Real-time PCR), which is to add a fluorescent group into a PCR reaction system and monitor the whole PCR process in Real time by utilizing fluorescent signal accumulation.
The microchip used in the present invention is a reaction region formed of a silica gel or an aluminum plate with a microreactor (the volume and mass of the microreactor depend on the type of microchip) covered with a protective film. The reaction area of the microchip is covered with a layer of mineral oil. Reagents are injected into the microreactor through a layer of mineral oil by a manual or automatic pipette with a gun head, so that cross contamination of detection samples and evaporation of a reaction system can be avoided.
The PCR reagent is freeze-dried on a microchip, and the fluorescent quantitative PCR technology of the Taqman probe is combined, so that the analysis of nucleic acid is that the microchip reads fluorescent signals generated by PCR products while thermal cycling. The microchip technology adopted by the invention is different from the PCR amplification using the plastic PCR tube as the carrier at present, but adopts the microchip of the metal carrier, and the reaction system and the sample are directly added on the metal carrier for PCR amplification. The heat conduction efficiency and the temperature rise and drop rate of the metal carrier are faster, and the reaction procedure time can be greatly shortened. The method has the characteristics of small reaction system, high automation degree, short reaction time, strong specificity, high sensitivity, strong repeatability, no need of low-temperature preservation of reaction reagents, capability of quantitative detection, small pollution possibility of microchip operation and the like. The product of the invention can rapidly identify and distinguish the monkey poxvirus, has simple operation, can finish from sample treatment to result analysis within 1 hour, namely, the result is obtained after 1 hour of clinical diagnosis, and the time of a molecular detection part is shortened to 30 minutes.
In conclusion, by adopting the technical scheme, the microchip fluorescence PCR reaction system capable of rapidly and effectively detecting the monkey pox virus is developed by designing the specific primers and the probes and optimizing the microchip freeze-drying condition, and the detection kit based on the method is prepared. A large number of experiments prove that the primer probe/kit/detection method has higher accuracy and excellent specificity compared with the conventional method, the minimum detection limit can reach 1copies/uL, the sensitivity is higher, meanwhile, the intra-batch variation coefficient of the monkey pox virus is between 0.29 and 0.92 percent, the inter-batch variation coefficient is between 0.36 and 1.01 percent, and the repeatability is excellent.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the specific detection of a monkey pox lyophilized microchip fluorescent PCR detection kit. In the figure, 1: monkey poxvirus; 2: measles virus; 3: varicella zoster virus; 4: herpes simplex virus type 1; 5: herpes simplex virus type 2; 6: human herpes type 6 virus; 7: EB virus; 8: negative control.
FIG. 2 is a sensitive assay of a monkey pox lyophilized microchip fluorescent PCR detection kit. In the figure, 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 cobies/uL; 9: nuclease-free water.
FIG. 3 is a standard curve of sensitivity detection for a monkey pox lyophilized microchip fluorescent PCR detection kit.
FIG. 4 is a sensitivity test of a commercially available product 1 kit. In the figure, 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 co-pins/uL; 9: nuclease-free water.
FIG. 5 is a sensitivity test standard curve of a commercially available product 1 kit.
FIG. 6 is a sensitivity test of a commercial product 2 kit. In the figure, 1: 1X 10 6 copies/uL；2：1×10 5 copies/uL；3：1×10 4 copies/uL；4：1×10 3 copies/uL；5：1×10 2 copies/uL；6：1×10 1 copies/uL；7：1copies/uL；8：10 -1 co-pins/uL; 9: nuclease-free water.
FIG. 7 is a sensitivity test standard curve of a commercially available product 2 kit.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental example 1 establishment of monkey pox lyophilized microchip fluorescence PCR detection method and determination of optimal condition
1. Design of primers and Taqman-MGB probes
According to the published monkey pox virus genes in China, specific conserved sequences of the monkey pox viruses are found, and a plurality of pairs of primers and probes are designed. Finally, a group of optimal primers and Taqman-MGB probes are determined through comparison and screening, and specific sequences are shown in Table 1:
TABLE 1 multiple primer and probe sequence information
Wherein the 5' end of the Taqman probe of the monkey poxvirus is marked with a FAM fluorescent reporter group; the 3' ends are marked with MGB quenching fluorescent groups. The reason for the quenching of the fluorophore to select MGB is that the TaqMan-MGB probe has the following advantages compared with the conventional TaqMan-TAMRA probe: (1) Increasing the TM value-15 bases on average increases 18 ℃, which can shorten the length of the probe, especially greatly facilitates the design of sequences with high AT content, and increases the TM value difference between paired and unpaired templates. (2) The signal to noise ratio is improved-because the 3' end quenching group of the probe is a fluorescent group which does not emit light, and the position of the fluorescent group is closer to that of the reporter group in space, the experimental result is more accurate, and the resolution ratio is higher.
2. Plasmid preparation
The gene plasmid of monkey pox virus, measles virus, varicella zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpes 6 virus and EB virus is synthesized by Huada gene technology service Co. Respectively dilute to 5 multiplied by 10 7 The copies/mL was placed at-20℃for further use.
3. Screening of optimal primer/Probe combinations
(1) The primer/probe specificity test comprises the steps of diluting three groups of primers/probes according to a specification, preparing primer/probe mixed solution, adding the mixed solution into a preliminarily planned 36 mu l reaction system, detecting 6 parts of specific quality control substances, comparing reaction results, and screening out the specific optimal primer/probe combination.
(2) Primer/probe sensitivity test gradient dilution samples (1X 10) of three sets of primer/probe pair sensitivity property controls 6 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 -1 Copy/. Mu.L), comparing the reaction results, and screening out the primer/probe combination with optimal sensitivity.
4. Freeze-dried microchip preparation
The PCR system formulation of the lyophilized microchip was performed according to Table 2.
TABLE 2PCR System
The whole lyophilized microchip loading well of the AriaYSB microchip (pegjingesen biosciences) for fluorescent quantitative PCR was 30, and the above-described fluorescent quantitative PCR system (containing 1 set of primer probes for monkey pox virus) was added at 1.2 μl per microchip well.
The microchip coated with the PCR reagent was frozen for 1 hour at-80 ℃. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
5. Primer/probe screening results
(1) The test results of the 3-group primer/probe specificity test result are shown in Table 3, and the 1 st group primer/probe (MPV-F/MPV-R/MPV-P), the 2 nd group primer/probe (MPV-F1/MPV-R1/MPV-P1) and the 3 rd group primer/probe (MPV-F2/MPV-R2/MPV-P2) have better specificity and do not have specific reaction on 6 nucleic acid samples such as measles virus, varicella zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpes type 6 virus, EB virus and the like and negative control (no nuclease water).
TABLE 3 primer/probe specificity test results
Note that: "+" indicates positive results; "-" indicates that the result is negative; "Unde" means that no fluorescent signal is detected.
(2) The test results of the 3-set primer/probe sensitivity test are shown in Table 4, and 1 copy/. Mu.L of nucleic acid sample can be detected by both the 1 st and 3 rd sets of primer/probes, but the Ct value of the 1 st set of primer/probe (MPV-F/MPV-R/MPV-P) is lower than that of the 3 rd set, and 1X 10 can be detected by the 2 nd set of primer/probe 2 Copy/. Mu.L of nucleic acid sample. Therefore, the group 1 primer/probe (MPV-F/MPV-R/MPV-P) was the most sensitive.
TABLE 4 primer/probe sensitivity test results
Note that: "+" indicates positive results; "-" indicates that the result is negative; "Unde" means that no fluorescent signal is detected.
(3) Primer/probe screening nodules
From the result of the primer/probe specificity experiment, the 3 groups of primers have better specificity and have no difference; from the results of the primer/probe sensitivity experiment, the 1 st primer/probe (MPV-F/MPV-R/MPV-P) sensitivity was good. The best primer/probe combination was considered to be MPV-F/MPV-R/MPV-P by combining the above experimental results.
Example 2 monkey pox lyophilized microchip fluorescent PCR detection kit specificity verification
After adding 24. Mu.L nuclease-free water to 10 Xbuffer dilution and shaking and mixing, 0.6. Mu.L mineral oil was added to each well, and 600. Mu.L mineral oil was added to the microchip surface, after which all wells were covered. Then, 0.6. Mu.L of gene plasmids of monkey pox virus, measles virus, varicella zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpes 6 virus and EB virus were added respectively, and 0.6. Mu.L of nuclease-free water was added to the final well as a negative control.
The conditions for the fluorescent PCR reaction of the lyophilized microchip were as follows: 45 ℃ for 5min, which is the first circulation; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
The experimental results are shown in fig. 1, the freeze-dried microchip is coated with a monkey pox virus single fluorescent PCR reagent, and the results in fig. 1 show that the monkey pox virus channel detection monkey pox virus plasmid is positive amplified, the Ct value is 22.84, and an amplification curve exists. The measles virus, varicella zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpes type 6, EB virus and negative control samples have no non-specific amplification, and the obtained results completely coincide with expectations. As can be seen from the amplification curves of all samples, the curve overlap is better at the early stages of amplification, especially around the fluorescence threshold (thresh old).
Example 3 monkey pox virus lyophilized microchip fluorescent PCR detection kit sensitivity verification and comparison with conventional commercially available fluorescent PCR reagents
The concentration was 1X 10 6 The copes/uL monkey poxvirus plasmid was subjected to 10-fold gradient dilution. Taking 1×10 6 copies/μL～10 -1 The sensitivity detection of lyophilized microchip fluorescent PCR reagents and conventional commercial fluorescent PCR reagents was performed using the plasmids of the monkey poxviruses at each gradient concentration of copies/. Mu.L as templates and nuclease-free water as negative control.
A lyophilized microchip fluorescent PCR system was prepared as in example 1, and after adding 24. Mu.L nuclease-free water to a 10 Xbuffer dilution, shaking and mixing, 0.6. Mu.L mineral oil was added to each well, and after all wells were covered with the microchip, 600. Mu.L mineral oil was added to the microchip surface. 1X 10 of each of the above-mentioned substances was added to the wells 6 copies/μL～10 -1 The concentration of each gradient of the papova plasmid was copies/. Mu.L, and 0.6. Mu.L of nuclease-free water was added to the final 1 well as a negative control, the sensitivity test results are shown in FIG. 2, and the sensitivity standard curve is shown in FIG. 3.
The conditions for the fluorescent PCR reaction of the lyophilized microchip were as follows: 45 ℃ for 5min, which is the first circulation; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, with fluorescence signal detection at the end of extension of each cycle.
Conventional commercial product 1 reaction procedure is as follows: the temperature is 50 ℃ for 15min, and the cycle is the first step; the temperature is 95 ℃ for 15min, and the cycle is the second step; 94℃for 10s and 55℃for 40s, 40 cycles of the third step, each cycle of which was extended to the end, were subjected to fluorescent signal detection.
Conventional commercial product 2 reaction procedure is as follows: the temperature is 50 ℃ for 10min, and the cycle is the first step; the temperature is 95 ℃ for 5min, and the second step of circulation is performed; 94℃ 15s and 55℃45s, 40 cycles of the third step, each cycle of which was extended to the end, were subjected to fluorescent signal detection.
The results in Table 5 show that the minimum detection sample concentration of the freeze-dried microchip fluorescence PCR is 1copies/uL and the Ct value is 37.39 due to the optimized PCR system, so that the minimum detection requirement can be greatly met by the reaction cycle number 40. Conventional commercial product 1 minimum detection sample concentration 1×10 1 The copies/uL, ct value is 38.23. The result of the sensitivity test of the conventional commercial kit product 1 is shown in FIG. 4, and the sensitivity standard curve is shown in FIG. 5.
Conventional commercial product 2 minimum detection sample concentration 1×10 1 The copies/uL, ct value is 38.13. The result of the sensitivity test of the conventional commercial kit product 2 is shown in FIG. 6, and the sensitivity standard curve is shown in FIG. 7. The results show that the sensitivity of the fluorescent PCR detection of the freeze-dried microchip is higher than that of the conventional commercial products.
TABLE 5 sensitivity assay for kits
| Sample name | Freeze-dried microchip Ct value | Commercial product 1Ct value | Commercial |
| 1×10 6 copies/uL | 19.42 | 22.95 | 22.96 |
| 1×10 5 copies/uL | 23.91 | 25.47 | 25.54 |
| 1×10 4 copies/uL | 27.63 | 28.85 | 28.37 |
| 1×10 3 copies/uL | 30.21 | 32.14 | 32.03 |
| 1×10 2 copies/uL | 32.51 | 35.13 | 35.03 |
| 1×10 1 copies/uL | 34.86 | 38.23 | 38.13 |
| 1copies/uL | 37.39 | Without any means for | Without any means for |
| 10 -1 copies/uL | Without any means for | Without any means for | Without any means for |
| Negative control | Without any means for | Without any means for | Without any means for |
The fluorescent PCR reaction system of the freeze-dried microchip is 1.2 mu L, the fluorescent PC R system of the conventional commercial product is 25 mu L, and the fluorescent PCR reaction system of the freeze-dried microchip is reduced by more than 20 times. The running time of the fluorescent PCR reaction of the freeze-dried microchip is less than 30 minutes, the actual running time of a conventional commercial product is 60-90 minutes, and the running time is shortened by 2/3.
Example 4 preparation of monkey pox lyophilized microchip fluorescence PCR detection kit and detection of reproducibility
1. Preparation of the kit:
preparation of lyophilized microchip:
the preparation of the lyophilized microchip PCR system was carried out according to the reaction system as shown in Table 6:
table 6 freeze-dried microchip PCR reaction system
The whole lyophilized microchip loading well of the AriaYSB microchip (pegjingesen biosciences) for fluorescent quantitative PCR was 30, and the above-described fluorescent quantitative PCR system (containing 1 set of primer probes for monkey pox virus) was added at 1.2 μl per microchip well.
The microchip coated with the PCR reagent was frozen for 1 hour at-80 ℃. The equipment freeze-drying conditions are as follows: in the pre-freezing stage, the temperature of the partition plate is reduced to-55 ℃, the retention time is 1h during pre-freezing, and then the equipment is vacuumized and kept for freeze drying for 1h; and in the desorption drying stage, the temperature of the separator is increased to-25 ℃ for 1h, then the temperature of the separator is increased to 37 ℃ and kept for 2h, and finally the temperature of the separator is reduced to 25 ℃ and kept for 1h.
Reagent 1: dilution Taq Buffer (10×) (Thermo Scientific, cat# B650060), 6 μl;
reagent 2: mineral oil (Sangon Biotech, cat. A630217) 1mL;
reagent 3: 30. Mu.L of positive control (monkey poxvirus positive plasmid);
reagent 4: negative control (nuclease free water) 30 μl;
reagent 5: nuclease-free water 50 μl.
2. Repetitive analysis of kits
3 samples of known positives were selected for each of the intra-batch and inter-batch replicates. Repeated detection in batch: 3 known positive samples were run in the same batch of experiments, with 3 replicates per sample. Experiments were repeated between batches: 3 known positive samples were tested in batches, each sample was tested separately, and each sample was set up with 3 replicates.
The fluorescence PCR reaction system of each freeze-dried microchip is 1.2 mu L: before using the reagent 1 (Taq Buffer), 24. Mu.L of the reagent 5 (no nuclease water) is required to be sucked into the reagent 1 (Taq Buffer) and mixed evenly by shaking, and then 0.6. Mu.L of the reagent is added into each well. 600. Mu.L of reagent 2 (mineral oil) was added to the microchip surface, after which it covered all of the wells. Each well was added with 0.6. Mu.L of each of the positive sample, reagent 3 (positive control) or reagent 4 (negative control).
The conditions for the fluorescent PCR reaction of the lyophilized microchip were as follows: 45 ℃ for 5min, which is the first circulation; 95 ℃ for 1min, which is the second circulation; 95℃for 5s and 60℃for 15s, 40 cycles of the third step, which was followed by fluorescence signal detection at the end of extension of each cycle, and recording of the experimental results.
As can be seen from the detection results in Table 7, the in-batch variation coefficient of the monkey poxvirus is between 0.29% and 0.92%, and the in-batch variation coefficient is between 0.36% and 1.01%, which indicates that the kit has good repeatability.
Table 7 kit reproducibility assay
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The primer probe combination is characterized in that the primers are 5 '-TCTTCCGTCAAT GTCTACACAG-3' and 5 '-GGATGCTGATACACGGCCT-3',
the probe is as follows: 5 '-TAATAACGGTGACAGGGTTAAC-3'.
2. A fluorescent lyophilized microchip for detecting a monkey pox virus, wherein a fluorescent PCR reaction system for detecting a monkey pox virus is immobilized on the fluorescent lyophilized microchip by lyophilization; the fluorescent PCR reaction system comprises the primer probe combination of claim 1.
3. The fluorescent lyophilized microchip according to claim 2,
the fluorescent PCR reaction system further comprises: DNA polymerase, dNTP and Mg 2+ Trehalose, tris-Cl and sterilized deionized water; the fluorescent PCR reaction system in each hole is 1-1.2 mu L.
4. The fluorescent lyophilized microchip according to claim 2,
the lyophilization comprises: placing the microchip provided with the fluorescent PCR reaction system in a temperature of minus 80 ℃ to minus 96 ℃ for freezing for 1 to 1.2 hours, and then performing equipment freeze-drying;
the apparatus lyophilization comprises: the method comprises a pre-freezing stage, a device vacuumizing stage, a freeze drying stage and a resolution drying stage, wherein the resolution drying stage comprises the following steps: raising the temperature to-25 to-20 ℃ and keeping for 1-1.5h, then raising the temperature of the separator to 30-37 ℃ and keeping for 1.5-2h, and finally lowering the temperature of the separator to 20-25 ℃ and keeping for 1-1.5h.
5. The fluorescent lyophilized microchip according to claim 2, wherein the sample addition holes on the fluorescent lyophilized microchip are 16, 20, 24, 30 or 48; the bottom structure, shape and size of the fluorescent freeze-dried microchip are matched with the sample adding plate of the PCR instrument.
6. A kit comprising the fluorescent lyophilized microchip according to any one of claims 2 to 5; the kit also comprises a diluent and mineral oil, wherein the diluent is dripped into a sample adding hole of the freeze-dried microchip, and then the freeze-dried microchip is subjected to fluorescent PCR amplification; the mineral oil is used to close the loading wells on the lyophilized microchip.
7. The kit of claim 6, further comprising: positive plasmid of monkey poxvirus was used as positive control; nuclease-free water served as a negative control.
8. Use of the primer probe combination of claim 1 or the fluorescent lyophilized microchip of any one of claims 2 to 5 or the kit of any one of claims 6 to 7 for detecting monkey pox virus.
9. A fluorescent PCR assay for identifying monkey poxviruses, characterized in that a sample to be tested is subjected to fluorescent PCR assay using the kit of any one of claims 6-7.
10. The method according to claim 9, wherein the reaction procedure of the fluorescent quantitative PCR comprises: the temperature is between 40 and 45 ℃ for 5 to 6 minutes, and the cycle is the first step; the temperature is between 90 and 95 ℃ for 1 to 2 minutes, and the second step of circulation is adopted; and (3) detecting fluorescent signals after the extension is finished in the third step of 38-40 cycles at the temperature of 90-95 ℃ for 5-6 s and at the temperature of 55-60 ℃ for 15-20 s.
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| WO2020124050A1 (en) * | 2018-12-13 | 2020-06-18 | The Broad Institute, Inc. | Tiled assays using crispr-cas based detection |
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| CN101407850A (en) * | 2008-11-20 | 2009-04-15 | 江苏出入境检验检疫局动植物与食品检测中心 | PCR fast detecting reagent kit and detecting method for monkeypox virus |
| WO2020124050A1 (en) * | 2018-12-13 | 2020-06-18 | The Broad Institute, Inc. | Tiled assays using crispr-cas based detection |
| CN111334608A (en) * | 2020-02-19 | 2020-06-26 | 北京亿森宝生物科技有限公司 | Dual-fluorescence freeze-drying microchip, kit and method for detecting novel coronavirus 2019-nCoV |
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