CN115044688A - Freeze-dried PCR reagent and kit for helicobacter pylori nucleic acid detection - Google Patents
Freeze-dried PCR reagent and kit for helicobacter pylori nucleic acid detection Download PDFInfo
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Abstract
The invention provides a freeze-dried PCR reagent and a kit for detecting helicobacter pylori nucleic acid, and particularly relates to a PCR amplification reagent (a primer and probe combination) with extremely high sensitivity for detecting the helicobacter pylori nucleic acid, which is obtained through a large amount of researches, and a freeze-dried system suitable for the PCR amplification reagent is further obtained through the researches on the freeze-dried reagent, so that the freeze-dried PCR reagent is prepared and obtained, and the problems of complicated preparation process, larger preparation error, poor reaction repeatability and higher product storage and transportation cost of a fluorescent PCR reaction reagent are solved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a freeze-dried PCR reagent and a kit for helicobacter pylori nucleic acid detection.
Background
Helicobacter Pylori (HP) is a gram-negative, microaerophilic, campylobacter bacterium that colonizes the human stomach. Helicobacter pylori infection is closely related to the occurrence and development of chronic atrophic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, gastric cancer and the like, and thus has attracted extensive clinical attention.
Most adult Hp infections are acquired in childhood, mostly in early childhood, and are generally difficult to spontaneously clear after infection, resulting in life-long infections. Therefore, screening and therapeutic research on Hp are very important for reducing the infection rate of Hp and improving the radical cure rate of Hp.
Currently, there are several approaches for Hp screening: 1. and (3) isolation culture identification: after the helicobacter pylori is cultured into bacteria, the bacteria are identified by biochemical reaction. Because the culture of the helicobacter pylori needs microaerophilic conditions and has strict requirements on nutritional conditions, the detection rate is extremely low, the method is not easy to popularize as a conventional diagnosis means, and the culture of the helicobacter pylori needs a certain time, so that the rapid diagnosis is not facilitated. 2. Urease-dependent test (urea breath test): the clinical application is wide, and the method is divided into a 13C breath test and a 14C breath test according to different markers. The defects are high cost, easy influence of bacteriostatic drugs and acid-inhibiting drugs and low sensitivity. 3. RUT and immunohistochemical staining, wherein the two methods need to take the gastric mucosa under a gastroscope and carry out staining analysis on a sample, and the method is obviously influenced by the loading capacity of helicobacter pylori, is complicated to operate, is time-consuming, is not suitable for detecting a sample with large flux, brings great pain to patients, and is especially suitable for the old and children. 4. And (3) immunological examination: detecting antibodies (IgG) in serum or saliva and urine or directly detecting antigen components of helicobacter pylori in feces. The sensitivity is very low and does not reflect symptomatic infection. 5. Nucleic acid analysis method: including sequencing, PCR, oligonucleotide probe hybridization, etc., but these detection methods have few detection sites, low specificity, low flux, high cost and no quantitative analysis.
The specific primer and probe for detecting helicobacter pylori are designed by a fluorescence PCR method, helicobacter pylori nucleic acid is directly detected, the sensitivity is high, the specificity is good, the operation is simple, and the helicobacter pylori can be detected in the same reaction and quantitatively analyzed by a multiple fluorescence PCR technology. The requirements for the fluorescent PCR reaction include: the fluorescent PCR reaction buffer system, the primers, the probes, the Taq enzyme and the dNTP need to be transported and stored at low temperature to avoid the change of the characters of the fluorescent PCR reaction buffer system and influence the using effect, the preparation process is complicated, the preparation error is large, the reaction repeatability is poor, and the product storage and transportation cost is high.
Disclosure of Invention
The invention aims to provide a freeze-dried PCR reagent for detecting helicobacter pylori nucleic acid, which aims to solve the problems of complicated preparation process, large preparation error, poor reaction repeatability and high product storage and transportation cost of a fluorescent PCR reaction reagent.
In a first aspect of the invention, a freeze-dried PCR reagent for helicobacter pylori nucleic acid detection is provided, and comprises an upstream primer, a downstream primer, a first probe and a second probe; wherein the sequence of the upstream primer is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.2, the sequence of the first probe is shown as SEQ ID NO.3, and the sequence of the second probe is shown as SEQ ID NO. 4.
In another preferred embodiment, two ends of the first probe and the second probe are respectively labeled with a fluorescent group and a quenching group; preferably, the fluorescent group is selected from the group consisting of: FAM, VIC, CY5, and Texas Red; the quenching group is selected from the group consisting of: BHQ1, BHQ2, BHQ3, and TAMRA.
In another preferred embodiment, the lyophilized PCR reagents further comprise a lyoprotectant.
In another preferred embodiment, the lyoprotectant includes one or more components selected from the group consisting of: trehalose, sucrose, bovine serum albumin, raffinose, dextran, glycerol, DTT, Proclin 300, formamide, and hydroxypropyl-beta-cyclodextrin.
In another preferred embodiment, the lyoprotectant consists of trehalose, raffinose, and sucrose.
In another preferred embodiment, the lyoprotectant includes:
2-10 parts by weight of trehalose,
2-10 parts by weight of raffinose, and
2-10 parts of sucrose.
In another preferred embodiment, the lyoprotectant includes:
5 parts by weight of trehalose, and a preparation method thereof,
5 parts by weight of raffinose, and
5 parts of cane sugar.
In another preferred embodiment, the lyophilized PCR reagents further comprise one or more components selected from the group consisting of: taq DNA polymerase, magnesium ions, potassium chloride, a non-ionic detergent, and Tris-HCL.
In another preferred example, when the lyophilized PCR reagent is used to prepare a PCR reaction system, the contents of the components in the PCR reaction system are:
the volume of the PCR reaction system was 25. mu.l.
In another preferred example, when the lyophilized PCR reagent is used to prepare a PCR reaction system, the contents of the components in the PCR reaction system are:
the volume of the PCR reaction system was 25. mu.l.
Further, the non-ionic detergent comprises one or more components selected from the group consisting of: triton X-100, Tween 20, and NP 40.
Further, the non-ionic detergent is triton X-100.
Further, the lyophilized PCR reagents were prepared by the following lyophilization procedure: 2.5h at 50 ℃ below zero; -50 ℃, 0.2mbar, 5 h; 0.2mbar at 0 ℃ for 0.5 h; 0.2mbar at 10 ℃ for 0.5 h; 20 ℃, 0.2mbar, 1 h; 20 ℃ and 2 h.
In a second aspect of the present invention, there is provided a kit for helicobacter pylori nucleic acid detection, the kit comprising the lyophilized PCR reagent according to the first aspect of the present invention.
In a third aspect of the present invention, there is provided a method for preparing a lyophilized PCR reagent for helicobacter pylori nucleic acid detection, the method comprising the steps of:
(1) preparation of liquid PCR reagent
The liquid PCR reagent comprises the following components in each 25 mul of liquid PCR reagent:
(2) freeze-drying
Lyophilizing the liquid PCR reagent provided in step (1), thereby obtaining the lyophilized PCR reagent.
In another preferred example, in the step (2), the lyophilization procedure is as follows: 2.5h at 50 ℃ below zero; -50 ℃, 0.2mbar, 5 h; 0.2mbar at 0 ℃ for 0.5 h; 0.2mbar at 10 ℃ for 0.5 h; 20 ℃, 0.2mbar, 1 h; 20 ℃ and 2 h.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is an amplification curve of different primer probe systems;
FIG. 2 shows lyophilized powder forms prepared from different lyoprotectant formulations;
FIG. 3 shows a PCR assay of nucleic acid samples extracted from gastric mucosal tissue;
FIG. 4 shows a comparison of the performance of PCR reagents before and after lyophilization.
Detailed Description
The invention provides a freeze-dried PCR reagent and a kit for detecting helicobacter pylori nucleic acid, and particularly relates to a PCR amplification reagent (a primer and probe combination) with extremely high sensitivity for detecting the helicobacter pylori nucleic acid, which is obtained through a large amount of researches, and a freeze-dried system suitable for the PCR amplification reagent is further obtained through the researches on the freeze-dried reagent, so that the freeze-dried PCR reagent is prepared and obtained, and the problems of complicated preparation process, larger preparation error, poor reaction repeatability and higher product storage and transportation cost of a fluorescent PCR reaction reagent are solved.
In a preferred embodiment, the invention provides a freeze-dried PCR reagent for helicobacter pylori nucleic acid detection, which comprises Taq DNA polymerase, magnesium ions, potassium chloride, a non-ionic detergent, Tris-HCL, a freeze-drying protective agent, an oligonucleotide upstream primer, an oligonucleotide downstream primer and an oligonucleotide fluorescent probe, wherein the oligonucleotide upstream primer has a sequence of 5'-CGTAGGAGTCTGGACCGTGT-3' (SEQ ID NO: 1), the oligonucleotide downstream primer has a sequence of 5'-CATAGGTCATGTGCCTCTTAGTTTGG-3' (SEQ ID NO: 2), the oligonucleotide fluorescent probe has a sequence of 5'-AGCCATTGGAAACGATGATTAATACCAGATACTC-3' (SEQ ID NO: 3), and the oligonucleotide fluorescent probe has a sequence of 5'-CTCAGGCCGGATACCCGTCATAGC-3' (SEQ ID NO: 4).
Furthermore, two ends of the oligonucleotide fluorescent probe are respectively marked with a fluorescent group and a quenching group, the fluorescent group is one of FAM, VIC, CY5 and Texas Red or other fluorescent groups with light-emitting characteristics, and the quenching group is one of BHQ1, BHQ2, BHQ3 and TAMRA or other chemical groups with fluorescence quenching functions. Preferably, the oligonucleotide fluorescent probe sequence 1 and the oligonucleotide fluorescent probe sequence 2 are labeled with the same fluorophore (e.g., FAM). More preferably, the oligonucleotide fluorescent probe sequence 1 and the oligonucleotide fluorescent probe sequence 2 are labeled with the same fluorescence quenching group (e.g., BHQ 1).
Further, each tube of lyophilized PCR reagents comprises: 0.1-1 mu M oligonucleotide upstream primer, 0.1-1 mu M oligonucleotide downstream primer, 0.1-1 mu M oligonucleotide fluorescent probe 1, 0.1-1 mu M oligonucleotide fluorescent probe 2, 0.1-1 unit Taq DNA polymerase, 1-10% M/v of freeze-drying protective agent, 1-5 mM magnesium ion, 10-100mM potassium chloride, 0-1% (M/v) nonionic detergent and 10-100mM Tris-HCL.
Further, each tube of lyophilized PCR reagents comprises: 0.2 μ M oligonucleotide upstream primer, 0.2 μ M oligonucleotide downstream primer, 0.1 μ M oligonucleotide fluorescent probe 1, 0.1 μ M oligonucleotide fluorescent probe 2, 0.2 unit Taq DNA polymerase and cryoprotectant with M/v of 5%, 3mM magnesium ion, 40mM potassium chloride, 0.05% (M/v) non-ionic detergent, 50mM Tris-HCL.
Further, the freeze-drying protective agent is one or a mixture of more of trehalose, sucrose, bovine serum albumin, raffinose, dextran, glycerol, DTT, Proclin 300, formamide, hydroxypropyl-beta-cyclodextrin and the like.
Furthermore, the freeze-drying protective agent is 2% -10% of trehalose, 2% -10% of raffinose and 2% -10% of sucrose.
Further, the cryoprotectant is 5% trehalose, 5% raffinose, 5% sucrose.
Further, the non-ionic detergent is one or more of Triton X-100, Tween 20, NP40 and the like.
Further, the non-ionic detergent is Triton X-100.
Further, lyophilized PCR reagents for helicobacter pylori nucleic acid detection were prepared by the following lyophilization procedure: 2.5h at 50 ℃ below zero; -50 ℃, 0.2mbar, 5 h; 0.2mbar at 0 ℃ for 0.5 h; 0.2mbar at 10 ℃ for 0.5 h; 20 ℃, 0.2mbar, 1 h; 20 ℃ and 2 h.
The invention takes the conserved sequence of the helicobacter pylori 16s RNA gene as a target to design an oligonucleotide primer and an oligonucleotide fluorescent probe, can specifically amplify the helicobacter pylori 16s RNA target sequence, extracts nucleic acid from gastric mucosa tissue to identify the helicobacter pylori, and uses a double-probe method to improve the detection sensitivity and the positive detection rate.
According to the invention, a suitable formula of the freeze-drying protective agent is optimized and screened to prepare the freeze-drying PCR reagent, so that a fluorescent PCR reaction buffer system, an oligonucleotide primer, an oligonucleotide probe, Taq DNA polymerase and dNTP contained in the reagent can stably coexist at normal temperature or even high temperature, and the prepared freeze-drying PCR reagent still keeps the detection effect after being transported at normal temperature. The production, transportation and storage costs of the fluorescent PCR reagent are greatly reduced, the use and operation steps can be reduced, the stability of the reagent is improved, the shelf life is prolonged, the flexibility of the sample volume is increased, and the detection sensitivity is improved. Contributes to the global sale and transportation of the fluorescent PCR reagent for detecting the helicobacter pylori.
The main advantages of the invention are:
(1) the kit has extremely high detection sensitivity and positive detection rate.
(2) The invention optimizes and screens a proper freeze-drying protective agent formula, so that the prepared freeze-drying PCR reagent for detecting the helicobacter pylori nucleic acid has stable form, and the amplification performance of the freeze-drying reagent is not different from that of a liquid amplification reagent.
(3) The freeze-dried PCR reagent for detecting the helicobacter pylori nucleic acid, which is prepared by the invention, contains a fluorescent PCR reaction buffer system, an oligonucleotide primer, an oligonucleotide probe, Taq DNA polymerase and dNTP which can stably coexist at normal temperature and even high temperature, and the prepared freeze-dried PCR reagent still keeps the detection effect after being transported at normal temperature.
(4) The freeze-dried PCR reagent for detecting the helicobacter pylori nucleic acid, which is prepared by the invention, can greatly reduce the production, transportation and storage costs of the PCR reagent for detecting the helicobacter pylori nucleic acid, reduce the use and operation steps, improve the stability of the reagent, prolong the shelf life, increase the flexibility of the sample volume and improve the detection sensitivity. Contributes to the global sale and transportation of the fluorescent PCR reagent for detecting the helicobacter pylori.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
EXAMPLE 1 establishment of PCR amplification System
The single probe detection system was prepared according to the following table, two single probe detection systems were prepared separately: oligonucleotide fluorescent probe 1 and oligonucleotide fluorescent probe 2:
the dual probe detection system was formulated as follows:
| components | Dosage (mu L) | Final concentration |
| Oligonucleotide upstream primer (10. mu.M) | 0.5 | 0.2μM |
| Oligonucleotide downstream primer (10. mu.M) | 0.5 | 0.2μM |
| Oligonucleotide fluorescent Probe 1 (10. mu.M) | 0.25 | 0.1μM |
| Oligonucleotide fluorescent probe 2 (10. mu.M) | 0.25 | 0.1μM |
| Taq DNA polymerase (5 units) | 1 | 0.2 unit |
| Magnesium ion (100mM) | 0.75 | 3mM |
| Potassium chloride (1M) | 1 | 40mM |
| Nonionic detergent (1%) | 1.25 | 0.05％ |
| Tris-HCL(100mM) | 12.5 | 50mM |
| ddH 2 O | 2 | |
| |
20 |
And (3) completing system preparation, adding 5 mu L of template into each reaction hole, mixing to obtain a 25 mu L reaction system, and carrying out PCR amplification reaction according to the following procedures: 1 minute at 95 ℃; fluorescence was detected by FAM channel at 95 deg.C, 5 seconds, 60 deg.C, 30 seconds, 40 cycles.
As shown in fig. 1: the Ct value and the fluorescence value of the same sample detected by the single probe-oligonucleotide fluorescent probe 1 and the oligonucleotide fluorescent probe 2 are basically the same; the Ct value detected by the combination of the double-probe oligonucleotide fluorescent probe 1 and the oligonucleotide fluorescent probe 2 is small and the fluorescence value is high.
EXAMPLE 2 establishment of Freeze-drying System
Freeze-drying PCR reagent for detecting helicobacter pylori prepared by different freeze-drying protective agent formulas
The lyoprotectant formulation combinations are as follows:
respectively preparing a freeze-drying protective agent mother solution according to a freeze-drying protective agent formula:
formula 1: 50% (m/v) trehalose;
and (2) formula: 50% (m/v) (trehalose + sucrose);
and (3) formula: 50% (m/v) (trehalose + raffinose);
and (4) formula: 50% (m/v) (trehalose + sucrose + raffinose).
Wherein m/v means mass concentration, which indicates how much mass per unit volume, and has a unit of "kg/L".
The freeze-dried PCR reagent for detecting helicobacter pylori nucleic acid is prepared as follows:
| components | Dosage (mu L) | Final concentration |
| Oligonucleotide upstream primer (10. mu.M) | 0.5 | 0.2μM |
| Oligonucleotide downstream primer (10. mu.M) | 0.5 | 0.2μM |
| Oligonucleotide fluorescent probe 1 (10. mu.M) | 0.25 | 0.1μM |
| Oligonucleotide fluorescent probe 2 (10. mu.M) | 0.25 | 0.1μM |
| Taq DNA polymerase (5 units) | 1 | 2.5 units |
| Magnesium ion (100mM) | 0.75 | 3mM |
| Potassium chloride (1M) | 1 | 40mM |
| Nonionic detergent (1%) | 1.25 | 0.05％ |
| Tris-HCL(100mM) | 12.5 | 50mM |
| Freeze-drying protective agent mother liquor (50%) | 2.5 | 5％ |
| ddH 2 O | 4.5 | |
| Total | 25 |
The above reagents were mixed and mixed well to prepare a PCR reagent with a final reaction volume of 25. mu.l.
Preparing a mixed solution (25 mu l multiplied by N) with a required volume according to requirements, uniformly mixing, subpackaging into 8-row PCR tubes, putting into a freeze-drying machine without covering to prepare a freeze-dried PCR reagent, and performing the following procedures: 2.5h at 50 ℃ below zero; -50 ℃, 0.2mbar, 5 h; 0.2mbar at 0 ℃ for 0.5 h; 0.2mbar at 10 ℃ for 0.5 h; 20 ℃, 0.2mbar, 1 h; 20 ℃ and 2 h.
And after the freeze drying is finished, packaging the freeze-dried PCR reagent in a nitrogen environment to prepare the freeze-dried PCR reagent for detecting the helicobacter pylori nucleic acid.
The morphology, morphology retention time (in air), powder properties and redissolution of lyophilized PCR reagents prepared with each lyoprotectant formulation were compared as follows:
note: +: the re-dissolution time is long, and oscillation is needed; ++: the re-dissolving time is long, and oscillation is not needed; +++: adding water to dissolve.
The freeze-drying PCR reagent prepared by the freeze-drying protective agent formula 4 has good shape and long shape maintaining time, can be ground into powder without residual water, has good redissolution property, and can be used as a freeze-drying protective agent formula for preparing the freeze-drying PCR reagent for detecting the helicobacter pylori nucleic acid.
EXAMPLE 3 detection of helicobacter pylori
A pathological section of the gastric mucosa of a normal person and a helicobacter pylori infection were prepared, and DNA was extracted with FFPE DNA/RNA Kit (Jiangsukang, century Biotechnology Co., Ltd.) for future use.
The lyophilized PCR reagent used in this example was the lyophilized PCR reagent for helicobacter pylori nucleic acid detection prepared by the formulation 4 in example 2.
And (3) taking 3 tubes of freeze-dried PCR reagents, respectively adding 25 mu l of the extracted DNA sample or sterile water contrast, after re-dissolving, ensuring that the total volume is 25 mu l, and shaking and uniformly mixing to ensure that the freeze-dried powder is completely dissolved.
The PCR amplification reaction was performed as follows: 1 minute at 95 ℃; fluorescence was detected by FAM channel at 95 deg.C, 5 seconds, 60 deg.C, 30 seconds, 40 cycles.
As shown in fig. 3: the blank control and the negative control have no Ct to be detected, and the amplification curve is a horizontal amplification curve; the helicobacter pylori infection sample has a Ct value for detection, an amplification curve is a typical S amplification curve, and a primer probe design principle is combined, so that the freeze-dried PCR reagent for detecting the helicobacter pylori nucleic acid, which is prepared in the embodiment 2, can effectively and specifically detect the helicobacter pylori.
Example 4 comparison of PCR reagent assay Performance Change before and after lyophilization
The lyophilized PCR reagent used in this example was the lyophilized PCR reagent for helicobacter pylori nucleic acid detection prepared in example 2 using formulation 4, and the control reagent was a liquid PCR reagent before lyophilization.
The H.pylori specimens used in this example were DNA extracted from pathological sections of the H.pylori-infected gastric mucosa in example 3.
The detection method is the same as that of example 3, and the detection result is shown in fig. 4: the freeze-dried PCR reagent for detecting the helicobacter pylori nucleic acid is prepared by using 5% of trehalose, 5% of raffinose and 5% of sucrose as freeze-drying protective agents, and the detection performance of the PCR reagent is basically unchanged before and after freeze-drying.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Jiangsukang was a century Biotechnology GmbH
Jiangsu Jian diagnostics science and technology Limited
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<213> Helicobacter pylori (Helicobacter pylori)
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Claims (10)
1. A freeze-dried PCR reagent for detecting helicobacter pylori nucleic acid is characterized by comprising an upstream primer, a downstream primer, a first probe and a second probe; wherein the sequence of the upstream primer is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.2, the sequence of the first probe is shown as SEQ ID NO.3, and the sequence of the second probe is shown as SEQ ID NO. 4.
2. The lyophilized PCR reagent of claim 1, wherein the first probe and the second probe are labeled with a fluorophore and a quencher at both ends thereof, respectively; preferably, the fluorescent group is selected from the group consisting of: FAM, VIC, CY5, and Texas Red; the quenching group is selected from the group consisting of: BHQ1, BHQ2, BHQ3, and TAMRA.
3. The lyophilized PCR reagent of claim 1, wherein the lyophilized PCR reagent further comprises a lyoprotectant.
4. The lyophilized PCR reagent of claim 3, wherein the lyoprotectant includes one or more components selected from the group consisting of: trehalose, sucrose, bovine serum albumin, raffinose, dextran, glycerol, DTT, Proclin 300, formamide, and hydroxypropyl-beta-cyclodextrin.
5. The lyophilized PCR reagent of claim 4, wherein the lyoprotectant consists of trehalose, raffinose, and sucrose.
6. The lyophilized PCR reagent of claim 5, wherein the lyoprotectant comprises:
2-10 parts by weight of trehalose,
2-10 parts by weight of raffinose, and
2-10 parts of sucrose.
7. The lyophilized PCR reagent of claim 1, further comprising one or more components selected from the group consisting of: taq DNA polymerase, magnesium ions, potassium chloride, a non-ionic detergent, and Tris-HCL.
8. The lyophilized PCR reagent of claim 7, wherein the non-ionic detergent comprises one or more components selected from the group consisting of: triton X-100, Tween 20, and NP 40.
9. A kit for helicobacter pylori nucleic acid detection, comprising the lyophilized PCR reagent of claim 1.
10. A method for preparing a lyophilized PCR reagent for helicobacter pylori nucleic acid detection, the method comprising the steps of:
(1) preparation of liquid PCR reagent
The liquid PCR reagent comprises the following components in each 25 mul of liquid PCR reagent:
(2) freeze-drying
Lyophilizing the liquid PCR reagent provided in step (1), thereby obtaining the lyophilized PCR reagent;
the sequence of the upstream primer is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.2, the sequence of the first probe is shown as SEQ ID NO.3, and the sequence of the second probe is shown as SEQ ID NO. 4.
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| CN202210713445.9A Pending CN115044688A (en) | 2022-06-22 | 2022-06-22 | Freeze-dried PCR reagent and kit for helicobacter pylori nucleic acid detection |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093403A (en) * | 2019-03-19 | 2019-08-06 | 融智生物科技（青岛）有限公司 | The freeze drying protectant of fluorescent PCR reagent and the preparation method that chip is lyophilized |
| CN116554998A (en) * | 2023-06-09 | 2023-08-08 | 鲲鹏基因（北京）科技有限责任公司 | Kit for detecting candida |
-
2022
- 2022-06-22 CN CN202210713445.9A patent/CN115044688A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093403A (en) * | 2019-03-19 | 2019-08-06 | 融智生物科技（青岛）有限公司 | The freeze drying protectant of fluorescent PCR reagent and the preparation method that chip is lyophilized |
| CN116554998A (en) * | 2023-06-09 | 2023-08-08 | 鲲鹏基因（北京）科技有限责任公司 | Kit for detecting candida |
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