CN114182046A - Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit - Google Patents
Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention relates to the field of detection of microorganisms and nucleic acid genomes, in particular to a primer combination and a probe for detecting nucleic acid of a common human herpesvirus (EB virus) clinically by using an isothermal nucleic acid amplification technology. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
Description
Technical Field
The invention relates to the field of detection of microorganisms and nucleic acid genomes, in particular to a primer probe combination and a kit for detecting pathogen nucleic acid of human herpesvirus and application thereof.
Background
EBV (Epstein-Barr virus, EBV) is a double-stranded DNA virus in the human herpesvirus family and can cause a plurality of common clinical diseases, EBV infection is the main cause of infectious mononucleosis, is common in children and is also closely related to B lymphoma related diseases and nasopharyngeal carcinoma. Like other herpesviruses, EB virus infection can also cause nervous system infections, respiratory diseases, and the like. The EB virus also has latent property, often remains latent in human B lymphocytes after infecting a human body, and is easily reactivated to cause related diseases under the condition of low human immunity, particularly in immature newborns, HIV infected patients and patients receiving organ transplantation, and the EB virus seriously harms the life and health of the human beings.
Currently, clinical etiological identification of EB virus infection mainly includes immunological detection and molecular biological detection. The immunological detection is suitable for epidemiological investigation, but has the problems of long time consumption, unstable window period and the like. Although heterophilic agglutination tests can suggest EBV infection, it is often negative in children and affects clinical doctors' disease judgment. In recent years, Quantitative Polymerase chain reaction (qPCR) has gradually become the "gold standard" for pathogen nucleic acid detection, and provides early diagnosis and prevention and treatment based on the Quantitative detection of pathogen nucleic acid. However, the complexity of expensive and sophisticated equipment is difficult in basic applications, and it is necessary to develop a simple, fast, convenient and applicable method for detecting nucleic acids.
Disclosure of Invention
In view of this, the invention provides a pathogen nucleic acid detection primer probe combination of human herpesvirus (EB virus), a kit and application thereof. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe combination, which comprises a first primer probe combination and/or a second primer probe combination;
the first primer probe combination is an isothermal nucleic acid amplification system combination containing an exogenous internal reference, and comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 4; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which is at least 80% identical to the nucleotide sequence shown in (I), (II), (III) and/or (IV); and/or
The second primer probe combination is an isothermal nucleic acid amplification system combination containing an endogenous internal reference, and comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 5; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 6; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 7; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I), (II), (III) and/or (IV).
In some embodiments of the invention, the primer probe combination further comprises a target gene probe having:
(I) a nucleotide sequence shown as SEQ ID No. 3; or
A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in the (I), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I); or
(III) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I) and/or (II).
In some embodiments of the invention, the second primer probe combination is an isothermal nucleic acid amplification system combination containing an endogenous internal reference, further comprising:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 3; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I), (II), (III) and/or (IV).
Based on the research, the invention also provides application of the primer combination in preparing a reagent or a kit for detecting the EB virus. In some embodiments of the invention, the human herpesvirus is epstein barr virus. In some embodiments of the invention, the amplification temperature of the assay is 39 ℃ and the amplification time is 30 min.
In addition, the invention also provides a detection reagent for human herpesvirus, which comprises the primer probe combination and an acceptable auxiliary agent. In some embodiments of the invention, the human herpes virus is epstein barr virus.
More importantly, the invention also provides a detection kit for human herpesvirus, which comprises the primer probe combination and acceptable auxiliary agents or carriers. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
A Recombinase-mediated isothermal nucleic acid amplification technology (RAA) is a newly emerged isothermal nucleic acid amplification technology with independent intellectual property rights in China in recent years, and is widely applied to the field of pathogen nucleic acid detection and single nucleotide polymorphism detection. The internal quality control (IC) comprises an exogenous internal reference and an endogenous internal reference, which is an important system monitoring of the nucleic acid detection reaction, and the IC is introduced into the nucleic acid detection reaction system to monitor the whole reaction system in real time, so that false negative results caused by misoperation, improper temperature, incorrect reaction system mixture, poor enzyme activity or the presence of inhibiting substances in a sample matrix and the like are avoided. Exogenous internal reference is that a section of internal reference template is added into a reaction system or a specimen. The endogenous reference adopts human genome from a sample, different primers are used for respectively carrying out amplification detection on the target gene and the human operation gene, more importantly, the whole process of nucleic acid extraction result interpretation can be monitored in real time, not only is the amplification reaction monitored, but also the sample collection quality and the nucleic acid extraction quality can be explained.
The invention adopts two different forms of IC to introduce into RAA reaction system, realize the real-time nucleic acid detection and monitoring of EB virus. In particular to a primer combination and a probe for detecting nucleic acid of a common human herpesvirus (EB virus) clinically by utilizing an isothermal nucleic acid amplification technology. The primer combination and the probe provided by the invention have the advantages of good specificity and high sensitivity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIGS. 1(A) to 1(B) are graphs showing the analysis of the concentration of an exogenous internal reference in the EBV technique for isothermal amplification of nucleic acids containing the exogenous internal reference, in which the target gene is 100Copying/reacting. Wherein, 1:1, 2:2, 3: 4; 4: 10;
FIGS. 2(A) to 2(B) are graphs showing the analysis of the concentration of an exogenous internal reference in the isothermal nucleic acid amplification detection EBV technique with an exogenous internal reference, with 5 copies/reaction of a target gene; wherein, 1:1, 2:2, 3: 4; 4: 10;
FIGS. 3(A) to 3(B) are graphs showing the analysis of the concentration of an exogenous internal reference in the EBV technique for isothermal amplification of nucleic acids and detection containing the exogenous internal reference, in which the target gene is 101Copying/reacting; wherein, 1:1, 2:2, 3: 4; 4: 10;
FIGS. 4(A) to 4(B) are sensitivity analysis charts of the isothermal nucleic acid amplification detection EBV technique containing the exogenous internal control. Wherein, 1: negative, 2: 5,3: 101，4：102，5:103,6:104；
FIGS. 5(A) to 5(B) are specific analytical drawings showing the isothermal nucleic acid amplification detection EBV technique with an exogenous internal reference;
FIGS. 6(A) to 6(B) are graphs showing the sensitivity analysis of the isothermal nucleic acid amplification detection EBV technique with an endogenous reference at a concentration of 2X 104Copies/. mu.L. Wherein, the ratio is 1:104，2:103，3： 1024：101,5: 2,6: negative;
FIGS. 7(A) to 7(B) are graphs showing the sensitivity analysis of the isothermal nucleic acid amplification detection EBV technique with an endogenous reference at a concentration of 2X 102Copy/. mu.L; wherein, the ratio is 1:104，2:103，3：1024： 101,5: 2,6: negative;
FIGS. 8(A) to 8(B) are graphs showing sensitivity analysis of the isothermal nucleic acid amplification detection EBV technique with an endogenous reference at a concentration of 2 copies/. mu.L; wherein, the ratio is 1:104，2:103，3：1024： 101,5: 2,6: negative;
FIGS. 9(A) to 9(B) are specific analysis charts showing the isothermal nucleic acid amplification detection EBV technique with an endogenous reference;
note: all panels a represent the amplification map of the EBV target gene of the present invention (FAM fluorescence), and all panels B represent the corresponding internal reference DNA amplification map of panel a (HEX fluorescence); the X-axis represents time (min) and the Y-axis represents fluorescence (mv).
Detailed Description
The invention discloses a primer probe combination for detecting pathogen nucleic acid of human herpesvirus, a kit and application thereof, and can be realized by appropriately improving process parameters by referring to the contents in the text by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In order to achieve the above object, the inventors searched a conserved sequence with high homology based on the genome sequence of EB virus and the genome sequence of human (RNaseP), and designed a specific primer pair and a target probe suitable for isothermal nucleic acid amplification technology. The specific DNA sequences of two target genes are provided by comparison, and the fragment positions of the two target genes are respectively the 177631-177771 bp (EBV) of the sequence disclosed by the GenBank accession number M80571.1 and the 554-708 bp (RNaseP) of the sequence disclosed by the GenBank accession number U77664.1.
The invention provides a primer probe combination, which comprises a first primer probe combination and/or a second primer probe combination;
the first primer probe combination is an isothermal nucleic acid amplification system combination containing an exogenous internal reference, and comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 4; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which is at least 80% identical to the nucleotide sequence shown in (I), (II), (III) and/or (IV); and/or
The second primer probe combination is an isothermal nucleic acid amplification system combination containing an endogenous internal reference, and comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 5; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 6; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 7; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I), (II), (III) and/or (IV).
In some embodiments of the invention, the primer probe combination further comprises a target gene probe having:
(I) a nucleotide sequence shown as SEQ ID No. 3; or
A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in the (I), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I); or
(III) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I) and/or (II).
In some embodiments of the present invention, the combination of primer probes, the second primer probe combination, and the isothermal nucleic acid amplification system comprising an endogenous internal reference further comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 3; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I), (II), (III) and/or (IV).
Based on the research, the invention also provides application of the primer combination in preparing a reagent or a kit for detecting human herpes viruses. In some embodiments of the invention, the primer probe is combined with a primer probe and the human herpesvirus is epstein barr virus.
In the embodiment of the invention, the isothermal nucleic acid amplification system adopted by the invention is specifically configured as follows: buffer solution, freeze-dried powder, a target gene forward primer, a target gene reverse primer, a target gene probe, an internal reference forward primer, an internal reference reverse primer, an internal reference probe, an internal reference and target gene template, ribozyme-free water and magnesium acetate.
More importantly, the invention also provides a detection reagent for human herpesvirus, which comprises the primer probe combination and acceptable auxiliary agents. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
The invention also provides a detection kit for human herpesvirus (EB virus), which comprises the primer probe combination and an acceptable auxiliary agent or carrier. In some embodiments of the invention, the human herpesvirus is epstein barr virus.
The invention adopts two forms of internal reference. An exogenous internal reference (recombinant DNA) is composed of plant virus segment and target gene segment (EBV segment), in which the complementary region of EBV target gene probe (SEQ ID NO.3) is substituted by plant virus segment, and the other sequence positions are retained. Under such a condition, the exogenous internal reference DNA is added in advance to the isothermal nucleic acid amplification reaction system, and the EBV target gene and the exogenous internal reference use the same primer sequence and perform fluorescence detection using their respective probes. The other is an endogenous reference, the endogenous reference is a human genome DNA fragment, and in this case, the EBV target gene and the exogenous reference are amplified and detected by using respective primers and probes.
In some embodiments of the present invention, the amplification detection temperature is 39-42 ℃, preferably 39 ℃, and the amplification detection time is 15-30 min, preferably 30 min.
And (4) interpretation of results: the system is mixed and then placed in a fluorescence detector with FAM (target gene) and HEX (internal reference) channels. In the amplification time range, the curve amplification slope of the fluorescence detector is set to be greater than or equal to 20, and the fluorescence detector has an obvious amplification curve, so that the fluorescence detector can be judged to be positive.
Wherein, FAM and HEX are both positive, and the positive result is judged.
FAM is positive, HEX is negative, and the result is judged to be positive.
FAM is negative, HEX is positive, and the result is judged to be negative.
Both FAM and HEX were negative and judged as invalid results.
The invention provides a primer combination and a probe for detecting nucleic acids of two clinically common human herpesviruses pathogens by using an isothermal nucleic acid amplification technology. The primer combination and the probe provided by the invention have good specificity and high sensitivity.
In the primer probe combination and the kit for detecting the pathogen nucleic acid of the human herpesvirus and the application thereof, all the used raw materials and reagents are commercially available. The RAA amplification detection kit (fluorescence method) was purchased from Jiangsu Qitian GmbH.
The invention is further illustrated by the following examples:
example 1 Synthesis of primer Probe design for detection of EBV target Gene and internal reference in isothermal nucleic acid amplification technology, and construction of recombinant target Gene plasmid and internal reference plasmid
Downloading and comparing the whole EBV genome sequence with the genome sequence of RNaseP, and selecting a sequence with high conservation. Primers and probes were designed manually for the EBV BARF1 gene and the RPP38 gene according to the RAA primer and probe design principle. The length ranges of the primers and probes are 30-35bp and 46-52bp, respectively. The probe consists of an upstream oligonucleotide carrying 5 '-FAM (target gene probe) and 5' -HEX (IC probe), each linked to an adjacent downstream oligonucleotide by a THF spacer. The downstream oligonucleotide carries a C3 ' spacer (polymerase extension blocking group) at the 3 ' end and the specificity of the primers and probes, whose sequences are shown in SEQ ID No.1-SEQ ID No.7, was analyzed and evaluated using Oligo7.0 software and NCBI's Primer BLAST software. All primers and probes were synthesized by a professional company, primers were purified by PAGE, and probes were purified by HPLC. The three probes adopt double-labeled probes, the target gene probe adopts FAM, the IC probe adopts HEX, the quenching groups all adopt BHQ-1, and tetrahydrofuran is arranged between the two fluorescent groups.
The exogenous internal reference sequence is a selected EBV target gene sequence, and the target gene probe position is replaced by a plant virus sequence segment. The target probe position was replaced with the rosette sequence in the selected EBV genomic sequence. And entrusting professional companies to synthesize and construct and return target genes and two internal reference sheets for system establishment and optimization in subsequent inventions.
EBV target gene sequence 5 '-3': (as shown in SEQ ID No. 8)
ATGGCCAGGTTCATCGCTCAGCTCCTCCTGTTGGCCTCCTGTGTGGCCGC CGGCCAGGCTGTCACCGCTTTCTTGGGTGAGCGAGTCACCCTGACCTCC TACTGGAGGAGGGTGAGCCTCGGTCCAGAGATTGAGGTCAGCTGGTTTA AACTGGGCCCAGGAGAGGAGCAGGTGCTTATTGGGCGCATGCACCACG ATGTCATCTTTATAGAGTGGCCTTTCAGGGGCTTCTTTGATATCCACAGA AGTGCCAACACCTTCTTTTTAGTAGTCACCGCTGCCAACATCTCCCATGA CGGCAACTACCTGTGCCGCATGAAACTGGGCGAGACCGAGGTCACCAA GCAGGAACACCTGAGCGTGGTGAAGCCTCTAACGCTGTCTGTCCACTCC GAAAGGTCTCAGTTCCCAGACTTCTCTGTCCTTACTGTGACATGCACCG TGAATGCATTTCCCCATCCCCACGTCCAGTGGCTCATGCCCGAGGGCGT GGAGCCCGCACCAACTGCGGCAAATGGCGGTGTTATGAAGGAAAAGGA TGGGAGCCTCTCTGTTGCTGTTGACCTGTCACTTCCCAAGCCCTGGCAC CTGCCAGTGACCTGCGTTGGGAAAAATGACAAGGAGGAAGCCCACGGG GTTTATGTTTCTGGATACTTGTCGCAATAA
Exogenous internal reference sequence 5 '-3' (shown as SEQ ID No. 9)
ATGGCCAGGTTCATCGCTCAGCTCCTCCTGTTGGCCTCCTGTGTGGCCG CCGGCCAGGCTGTCACCGCTTTCTTGGGTGAGCGAGTCACCCTGACCTC CTACTGGAGGAGGGTGAGCCTCGGTCCAGAGATTGAGGTCAGCTGGTT TAAACTGGGCCCAGGAGAGGAGCAGGTGCTTATTGGGCGCATGCACCA CGATGTCATCTTTATAGAGTGGCCTTTCAGGGGCTTCTTTGATATCCAC AGAAGTGCCAACACCTTCTTTTTAGTAGTCACCGCTGCCAACATCTCCC ATGACGGCAACTACCTGTGCCGCATGAAACTGGGCGAGACCGAGGTCA CCAAGCAGGAACACCTGAGCGTGGTGAAGCCTCTAACGCTGTCTGTCC ACTCCGAAAGGTCTCAGTTCCCAGACTTCTCTGTCCTTACTGTGACATG CACCGTGAATGCATTTCCCCATCCCCACGTCCAGTGGCTCATGCCCGAG GGCGTGGAGCCCGCACCAACTGCGGCAAATGGCGGTGTTATGAAGGAA AAGGATGGGAGCCTCTCTGTTGCTGTTGACCTGTCACTTCCCAAGCCCT GGCACCTGCCAGTGACCTGCGTTGGGAAAAATGACAAGGAGGAAGCCC ACGGGGTTTATGTTTCTGGATACTTGTCGCAATAA
Endogenous reference sequence 5 '-3' (shown as SEQ ID No. 10)
TGGACTTCAGAAGATTGAAGATAAGAAGAAAAAGAACAAAACACCTTT TCTGAAAAAAGAAAGCAGAGAGAAATGCAGCATTGCTGTTGATATTAG TGATAATCTGAAGGAGAAGAAAACAGATGCTAAGCAGCAAGTGTCAG GGTGGACGCCTGCACACGTCAGGAAGCAGCTTGTCATTGGCGTTAACG AAGTTACCAGAGCCCTGGAAAGGAGGGAACTGCTGTTAGTTCTGGTGT GTAAATCAGTCAAGCCTGCCATGATCACCTCACACTTGATTCAGTTAAG CCTAAGCAGAAGTGTCCCTGCCTGTCAGGTCCCCCGGCTCAGTGAGAG AATCGCCCCCGTCATTGGCTTAAAATGTGTTCTAGCCTTGGCGTTCAAA AAGAACACCACTGACTTTGTGGACGAAGTAAGAGCCATCATCCCCAGA GTCCCCAGTTTAAGTGTACCATGGCTTCAAGACAGAATTGAAGATTCTG GGGAAAATTTAGAGACTGAACCTCTGGAAAGCCAAGACAGAGAGCTTT TGGACACTTCATTTGAAGATCTGTCAAAACCTAAGAGAAAGCTTGCTG ACGGTCGGCAGGCTTCTGTAACATTACAACC
Example 2 isothermal nucleic acid amplification detection with exogenous internal reference EBV technology System establishment, sensitivity analysis and specificity analysis, clinical evaluation
1. The primer probe (SEQ ID No.1-SEQ ID No.4) designed and synthesized in example 1 was used as the primer and probe. The template used was the EBV target gene DNA synthesized in example 1 and the exogenous reference DNA.
2. The reaction reagent adopts RAA fluorescence detection kit of Jiangsu Qitian, and the whole isothermal nucleic acid amplification system (50ul) is configured as follows: 25 mu L of buffer solution, a forward primer (shown as SEQ ID No.1, 420nM), a reverse primer (shown as SEQ ID No.2, 420nM), a target gene probe (shown as SEQ ID No.3, 120nM), an exogenous internal reference probe (shown as SEQ ID No.4, 120nM), exogenous internal reference DNA, 1 mu L of template and adjustable water volume without nuclease. The mixed reaction system is added into a reaction tube (single-chain binding protein 500 ng/. mu. L, DNA polymerase 90 ng/. mu.L, recombinase 400 ng/. mu.L, exonuclease 85 ng/. mu.L and UvsY protein 70 ng/. mu.L) which is pre-filled with freeze-dried powder, and finally magnesium acetate 14mM is added.
3. And (4) interpretation of results: after the system is mixed, the reaction tube is placed in a fluorescence detector with FAM (target gene) and HEX (internal reference) channels. In the amplification time range, the curve amplification slope of the fluorescence detector is set to be more than or equal to 20, and the fluorescence detector has an obvious amplification curve, so that the fluorescence detector can be judged to be positive. The amplification effect is mainly judged by the intensity of a fluorescence signal and the time of peak formation.
Wherein, FAM and HEX are both positive, and the positive result is judged.
FAM is positive, HEX is negative, and the result is judged to be positive.
FAM is negative, HEX is positive, and the result is judged to be negative.
And judging that both FAM and HEX are negative and are invalid, and recommending to detect again.
4. Confirmation of exogenous internal reference concentration
According to experience, when the exogenous internal reference DNA concentration is respectively 10 copies/muL, 4 copies/muL, 2 copies/muL and 1 copy/muL, the low-copy EBV target gene standard plasmids (1, 5 and 10 copies/reaction) are detected, so that the amplification of the exogenous internal reference can not influence the normal amplification of the target gene and the proper exogenous internal reference DNA concentration is confirmed.
The results show that when the copy number of the EBV target gene is 10 copies, the exogenous internal references with 4 different concentrations are judged to be positive, the normal amplification of the target gene is not influenced, and the amplification effect of the target gene has no obvious difference. As shown in fig. 3A-3B. When the copy number of the EBV target gene is 5 copies, the exogenous internal references with 4 different concentrations are judged to be positive, and the normal amplification of the target gene is not influenced. Wherein, in the presence of exogenous internal reference DNA concentration of 1 copy/. mu.L, the amplification effect (fluorescence value and time to peak) of the target gene (1 copy) is better than that of the target gene in the presence of 3 other exogenous internal reference DNA concentrations. As shown in fig. 3A-3B. When the copy number of the EBV target gene is 1 copy, the exogenous internal parameters with 4 different concentrations are judged to be positive, and the target gene is negative. As shown in fig. 3A-3B. The same amplification results can be obtained by repeating the above examples, and the repeatability is good. Therefore, the present invention obtained according to this example, the amplification of the internal reference did not affect the amplification of the target gene, and the amplification effect of the target gene was optimized. The final concentration of the appropriate exogenous internal control DNA chosen was 1 copy per microliter.
5. The isothermal nucleic acid amplification technique of the invention for detecting EBV, which contained an exogenous internal reference, was analyzed for sensitivity and specificity according to the system and exogenous internal reference DNA concentrations determined in this example.
Using the EBV target Gene template of real-time example 1 (10)4、103、102、101、5、100Copy/reaction) for sensitivity analysis. At an exogenous internal reference DNA concentration of 1 copy/. mu.L, amplification of the internal reference does not affect normal amplification of the target gene, and the sensitivity of the target gene is 5 copies/reaction. The above results were repeated with good reproducibility. The results are shown in FIGS. 4A-4B.
Clinical sample positive nucleic acids using other viruses collected in the present invention include EBV, human herpesvirus type 1, 2, 5, 6, human hepatitis B, human hepatitis C and West Nile viruses, and the human genome. The result shows that no cross reaction exists with other pathogens, which indicates that the method has good specificity. As shown in fig. 5A-5B.
Example 3 establishment of isothermal nucleic acid amplification detection EBV technology System containing endogenous reference, sensitivity analysis, specificity analysis
1. Different from example 2, the primer and probe used were the primer probes (SEQ ID Nos. 1 to 3 and 5 to 7) designed and synthesized in example 1. The template used was the EBV target gene DNA and the endogenous reference DNA constructed as in example 1.
2. The reaction reagent adopts RAA fluorescence detection kit of Jiangsu Qitian, and the whole isothermal nucleic acid amplification system (50ul) is configured as follows: 25 mu L of buffer solution, a target gene forward primer (shown as SEQ ID No.1, 420nM), a reverse primer (shown as SEQ ID No.2, 420nM), a target gene probe (shown as SEQ ID No.3, 120nM), an endogenous reference forward primer (shown as SEQ ID No.4, 120nM), an endogenous reference reverse primer (shown as SEQ ID No.4, 120nM), an endogenous reference probe (shown as SEQ ID No.4, 50nM), nuclease-free water and template, wherein the volumes of the buffer solution, the target gene forward primer (shown as SEQ ID No.1, 420nM), the endogenous reference forward primer, the endogenous reference reverse primer, the endogenous reference probe and the nuclease-free water and the template are adjustable. The mixed reaction system is added into a reaction tube (single-chain binding protein 500 ng/. mu. L, DNA polymerase 90 ng/. mu.L, recombinase 400 ng/. mu.L, exonuclease 85 ng/. mu.L and UvsY protein 70 ng/. mu.L) which is pre-filled with freeze-dried powder, and finally magnesium acetate 14mM is added.
3. And (4) interpretation of results: after the system is mixed, the reaction tube is placed in a fluorescence detector with FAM (target gene) and HEX (internal reference) channels. In the amplification time range, the curve amplification slope of the fluorescence detector is set to be more than or equal to 20, and the fluorescence detector has an obvious amplification curve, so that the fluorescence detector can be judged to be positive. The amplification effect is mainly judged by the intensity of a fluorescence signal and the time of peak formation.
Wherein, FAM and HEX are both positive, and the positive result is judged.
FAM is positive, HEX is negative, and the result is judged to be positive.
FAM is negative, HEX is positive, and the result is judged to be negative.
And judging that both FAM and HEX are negative and are invalid, and recommending to detect again.
4. The isothermal nucleic acid amplification technique of the present invention for detecting EBV comprising an exogenous internal control was analyzed for sensitivity and specificity according to the system defined in this example.
Empirically, the EBV target gene template of real-time example 1 (10) was used4、103、102、1012 copies/reaction), sensitivity analysis was performed in the presence of 3 different endogenous references. The results showed that the endogenous reference concentration was 2X 104Copy/. mu.L, 8 times of repeated detection results, and the sensitivity can reach 2 copies per reaction. The results are shown in FIGS. 6A-6B; the endogenous reference concentration is 2 × 102Copy/. mu.L, 8 times of repeated detection results, and the sensitivity can reach 2 copies per reaction. The results are shown in FIGS. 7A-7B; at an endogenous reference concentration ofThe sensitivity of the detection result is 2 copies/. mu.L, and the detection result is repeated for 8 times, and the sensitivity can reach 2 copies per reaction. The results are shown in FIGS. 8A-8B.
Clinical sample positive nucleic acids using other viruses collected in the present invention include EBV, human herpesvirus type 1, 2, 5, 6, human hepatitis B virus, human hepatitis C virus and West Nile virus. The result shows that no cross reaction exists with other pathogens, which indicates that the method has good specificity. As shown in fig. 9A-9B.
EXAMPLE 4 evaluation of clinical specimens
125 clinical samples of patients suspected of EBV infection were collected, DNA was extracted using Qiagen miniDNA Kit, and clinical samples were evaluated for both EBV IC-RAA methods.
1. The samples were subjected to DNA detection using commercial polymerase chain reaction quantification kit of EBV.
2. The reaction procedures are as follows: 93 ℃ for 2 min; 93 ℃, 45s, 55 ℃, 1min, 10 cycles; 93 ℃, 30s, 55 ℃, 45s, 30 cycles.
3. Reaction components: 42 μ of LPCR reaction, 3 μ of LTaq enzyme, 5 μ of LDNA.
4. And (4) interpretation of results: positive when CT is less than or equal to 30; CT > 30, negative.
5. The collected specimens were examined according to the isothermal nucleic acid amplification detection EBV technique system containing the exogenous internal reference established in example 2 and the isothermal nucleic acid amplification detection EBV technique system containing the endogenous internal reference established in example 3.
6. The interpretation of the results is referred to examples 2 and 3.
The results are shown in the following table:
TABLE 1
The collected clinical samples are evaluated by using the isothermal nucleic acid amplification detection EBV technical system containing the exogenous internal reference established in the embodiment 2 and the isothermal nucleic acid amplification detection EBV technical system containing the endogenous internal reference established in the embodiment 3 respectively, and the statistical comparison with the commercial quantitative PCR kit shows good consistency and good repeatability.
The clinical sample evaluation species of the two methods find that the internal references of the two different forms are detected as positive results, do not affect the target gene EBV amplification, and are suitable for the amplification monitoring of an RAA reaction system. In the RAA technology containing endogenous reference, the evaluation of clinical samples indirectly proves that the extraction effect of the sample nucleic acid DAN is good. The two different forms of internal references in the invention can be successfully applied to the RAA technology, and have good effect and good repeatability.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Feng Shishan
Primer and probe combination and kit for detecting pathogen nucleic acid of <120> human herpesvirus and application of primer and probe combination and kit
<130> MP21019590
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctacctgtgc cgcatgaaac tgggcgagac cga 33
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
catgtcacag taaggacaga gaagtctggg 30
<210> 3
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n(32)=i6famdt
<220>
<221> misc_feature
<222> (33)..(33)
<223> n(33)=idsp
<220>
<221> misc_feature
<222> (34)..(34)
<223> n(34)=ibhq1dt
<220>
<221> misc_feature
<222> (49)..(49)
<223> n(49)=c3-spacer
<400> 3
gaacacctga gcgtggtgaa gcctctaacg cnnnctgtcc actccgaan 49
<210> 4
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n(32)=hexdt
<220>
<221> misc_feature
<222> (34)..(34)
<223> n(34)=idsp
<220>
<221> misc_feature
<222> (36)..(36)
<223> n(36)=bhq-dt
<220>
<221> misc_feature
<222> (52)..(52)
<223> n(52)=c3-spacer
<400> 4
gtaaggtgct agactaaaat tgttgggact tngnanctct gaagtaaaag gn 52
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcttaaaatg tgttctagcc ttggcgttca 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cagaggttca gtctctaaat tttccccaga 30
<210> 7
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n(32)=i6famdt
<220>
<221> misc_feature
<222> (34)..(34)
<223> n(34)=idsp
<220>
<221> misc_feature
<222> (36)..(36)
<223> n(36)=ibhq1dt
<220>
<221> misc_feature
<222> (52)..(52)
<223> n(52)=c3-spacer
<400> 7
gtaaggtgct agactaaaat tgttgggact tngnanctct gaagtaaaag gn 52
<210> 8
<211> 666
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggccaggt tcatcgctca gctcctcctg ttggcctcct gtgtggccgc cggccaggct 60
gtcaccgctt tcttgggtga gcgagtcacc ctgacctcct actggaggag ggtgagcctc 120
ggtccagaga ttgaggtcag ctggtttaaa ctgggcccag gagaggagca ggtgcttatt 180
gggcgcatgc accacgatgt catctttata gagtggcctt tcaggggctt ctttgatatc 240
cacagaagtg ccaacacctt ctttttagta gtcaccgctg ccaacatctc ccatgacggc 300
aactacctgt gccgcatgaa actgggcgag accgaggtca ccaagcagga acacctgagc 360
gtggtgaagc ctctaacgct gtctgtccac tccgaaaggt ctcagttccc agacttctct 420
gtccttactg tgacatgcac cgtgaatgca tttccccatc cccacgtcca gtggctcatg 480
cccgagggcg tggagcccgc accaactgcg gcaaatggcg gtgttatgaa ggaaaaggat 540
gggagcctct ctgttgctgt tgacctgtca cttcccaagc cctggcacct gccagtgacc 600
tgcgttggga aaaatgacaa ggaggaagcc cacggggttt atgtttctgg atacttgtcg 660
caataa 666
<210> 9
<211> 666
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atggccaggt tcatcgctca gctcctcctg ttggcctcct gtgtggccgc cggccaggct 60
gtcaccgctt tcttgggtga gcgagtcacc ctgacctcct actggaggag ggtgagcctc 120
ggtccagaga ttgaggtcag ctggtttaaa ctgggcccag gagaggagca ggtgcttatt 180
gggcgcatgc accacgatgt catctttata gagtggcctt tcaggggctt ctttgatatc 240
cacagaagtg ccaacacctt ctttttagta gtcaccgctg ccaacatctc ccatgacggc 300
aactacctgt gccgcatgaa actgggcgag accgaggtca ccaagcagga acacctgagc 360
gtggtgaagc ctctaacgct gtctgtccac tccgaaaggt ctcagttccc agacttctct 420
gtccttactg tgacatgcac cgtgaatgca tttccccatc cccacgtcca gtggctcatg 480
cccgagggcg tggagcccgc accaactgcg gcaaatggcg gtgttatgaa ggaaaaggat 540
gggagcctct ctgttgctgt tgacctgtca cttcccaagc cctggcacct gccagtgacc 600
tgcgttggga aaaatgacaa ggaggaagcc cacggggttt atgtttctgg atacttgtcg 660
caataa 666
<210> 10
<211> 609
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tggacttcag aagattgaag ataagaagaa aaagaacaaa acaccttttc tgaaaaaaga 60
aagcagagag aaatgcagca ttgctgttga tattagtgat aatctgaagg agaagaaaac 120
agatgctaag cagcaagtgt cagggtggac gcctgcacac gtcaggaagc agcttgtcat 180
tggcgttaac gaagttacca gagccctgga aaggagggaa ctgctgttag ttctggtgtg 240
taaatcagtc aagcctgcca tgatcacctc acacttgatt cagttaagcc taagcagaag 300
tgtccctgcc tgtcaggtcc cccggctcag tgagagaatc gcccccgtca ttggcttaaa 360
atgtgttcta gccttggcgt tcaaaaagaa caccactgac tttgtggacg aagtaagagc 420
catcatcccc agagtcccca gtttaagtgt accatggctt caagacagaa ttgaagattc 480
tggggaaaat ttagagactg aacctctgga aagccaagac agagagcttt tggacacttc 540
atttgaagat ctgtcaaaac ctaagagaaa gcttgctgac ggtcggcagg cttctgtaac 600
attacaacc 609
Claims (10)
1. A primer probe combination, which is characterized by comprising a first primer probe combination and/or a second primer probe combination;
the first primer probe combination is an isothermal nucleic acid amplification system combination containing an exogenous internal reference, and comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 4; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which is at least 80% identical to the nucleotide sequence shown in (I), (II), (III) and/or (IV); and/or
The second primer probe combination is an isothermal nucleic acid amplification system combination containing an endogenous internal reference, and comprises:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 5; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 6; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 7; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I), (II), (III) and/or (IV).
2. The primer probe combination of claim 1, further comprising a target gene probe having:
(I) a nucleotide sequence shown as SEQ ID No. 3; or
A nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in the (I) and has the same or similar functions with the nucleotide sequence shown in the (I); or
(III) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I) and/or (II).
3. The primer probe combination of claim 1 or 2, wherein the second primer probe combination is an isothermal nucleic acid amplification system combination comprising an endogenous internal reference, further comprising:
(I) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and
(II) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; and
(III) the probe has a nucleotide sequence shown as SEQ ID No. 3; or
(IV) a nucleotide sequence which is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (I), (II) and/or (III) and has the same or similar functions with the nucleotide sequence shown in (I), (II) and/or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence shown in (I), (II), (III) and/or (IV).
4. Use of a primer combination according to any one of claims 1 to 3 for the preparation of a reagent or kit for the detection of epstein barr virus.
5. The use according to claim 4, wherein the human herpesvirus is Epstein-Barr virus.
6. The use of claim 5, wherein the detected amplification temperature is 39 ℃ and the amplification time is 30 min.
7. A reagent for the detection of human herpesvirus comprising the primer probe combination of any one of claims 1 to 3 and an acceptable auxiliary.
8. The detection reagent according to claim 7, wherein the human herpesvirus is epstein barr virus.
9. A kit for the detection of human herpesvirus comprising the primer probe combination of any one of claims 1 to 3 and an acceptable adjuvant or carrier.
10. The test kit of claim 9, wherein the human herpesvirus is epstein barr virus.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016011280A1 (en) * | 2014-07-16 | 2016-01-21 | Tangen Biosciences, Inc. | Isothermal methods for amplifying nucleic acid samples |
| CN110106285A (en) * | 2019-03-19 | 2019-08-09 | 中国疾病预防控制中心病毒病预防控制所 | A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection |
| CN111154913A (en) * | 2018-11-08 | 2020-05-15 | 中山大学 | Primer and crRNA for EB virus DNA detection and application thereof |
| CN113122660A (en) * | 2021-05-19 | 2021-07-16 | 冯志山 | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit |
-
2021
- 2021-08-18 CN CN202110949243.XA patent/CN114182046B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016011280A1 (en) * | 2014-07-16 | 2016-01-21 | Tangen Biosciences, Inc. | Isothermal methods for amplifying nucleic acid samples |
| CN111154913A (en) * | 2018-11-08 | 2020-05-15 | 中山大学 | Primer and crRNA for EB virus DNA detection and application thereof |
| CN110106285A (en) * | 2019-03-19 | 2019-08-09 | 中国疾病预防控制中心病毒病预防控制所 | A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection |
| CN113122660A (en) * | 2021-05-19 | 2021-07-16 | 冯志山 | Primer probe combination and kit for detecting pathogen nucleic acid of human herpesvirus and application of primer probe combination and kit |
Non-Patent Citations (3)
| Title |
|---|
| GAO YUAN: "Rapid Internal Control Reference Recombinase-Aided", 《BIOMED ENVIRON SCI》 * |
| JING‑YI LI: "Detection of low-load Epstein-Barr virus", 《AMB EXPRESS》 * |
| 何安娜: "人乳头瘤病毒6型和11型通...等温扩增技术检测方法的建立", 《新发传染病电子杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116516069A (en) * | 2023-05-10 | 2023-08-01 | 武汉大学 | RPA kit for rapidly detecting Epstein-Barr virus in cells |
| CN116516069B (en) * | 2023-05-10 | 2024-03-22 | 武汉大学 | RPA kit for rapidly detecting Epstein-Barr virus in cells |
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