CN114350858A - Primer probe composition for detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) viruses and application thereof - Google Patents
Primer probe composition for detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) viruses and application thereof Download PDFInfo
- Publication number
- CN114350858A CN114350858A CN202210118685.4A CN202210118685A CN114350858A CN 114350858 A CN114350858 A CN 114350858A CN 202210118685 A CN202210118685 A CN 202210118685A CN 114350858 A CN114350858 A CN 114350858A
- Authority
- CN
- China
- Prior art keywords
- hcmv
- ebv
- primer
- probe composition
- primer probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000701024 Human betaherpesvirus 5 Species 0.000 title claims abstract description 79
- 239000000523 sample Substances 0.000 title claims abstract description 79
- 239000000203 mixture Substances 0.000 title claims abstract description 54
- 241000701044 Human gammaherpesvirus 4 Species 0.000 title abstract description 63
- 241000700605 Viruses Species 0.000 title description 15
- -1 pplication Species 0.000 title description 2
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 239000013641 positive control Substances 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 230000002441 reversible effect Effects 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 241001112090 Pseudovirus Species 0.000 claims description 7
- 239000013642 negative control Substances 0.000 claims description 7
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 239000007850 fluorescent dye Substances 0.000 claims description 4
- 239000013612 plasmid Substances 0.000 claims description 4
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 3
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 3
- 239000011654 magnesium acetate Substances 0.000 claims description 3
- 229940069446 magnesium acetate Drugs 0.000 claims description 3
- 235000011285 magnesium acetate Nutrition 0.000 claims description 3
- 235000011056 potassium acetate Nutrition 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims 1
- 238000010791 quenching Methods 0.000 claims 1
- 230000000171 quenching effect Effects 0.000 claims 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical group C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000003759 clinical diagnosis Methods 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 23
- 208000015181 infectious disease Diseases 0.000 description 13
- 241001529453 unidentified herpesvirus Species 0.000 description 10
- 239000000126 substance Substances 0.000 description 9
- 230000003321 amplification Effects 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 5
- 230000029812 viral genome replication Effects 0.000 description 5
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 4
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 108091036078 conserved sequence Proteins 0.000 description 4
- 230000000405 serological effect Effects 0.000 description 4
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- AFWTZXXDGQBIKW-UHFFFAOYSA-N C14 surfactin Natural products CCCCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 AFWTZXXDGQBIKW-UHFFFAOYSA-N 0.000 description 2
- 241001502974 Human gammaherpesvirus 8 Species 0.000 description 2
- 101150089187 IE1 gene Proteins 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- NJGWOFRZMQRKHT-UHFFFAOYSA-N surfactin Natural products CC(C)CCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-UHFFFAOYSA-N 0.000 description 2
- NJGWOFRZMQRKHT-WGVNQGGSSA-N surfactin C Chemical compound CC(C)CCCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-WGVNQGGSSA-N 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- 230000006648 viral gene expression Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 101150059079 EBNA1 gene Proteins 0.000 description 1
- 206010015108 Epstein-Barr virus infection Diseases 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 101000758973 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 11.3 kDa protein in lys 3'region Proteins 0.000 description 1
- 101000652805 Homo sapiens Protein shisa-8 Proteins 0.000 description 1
- 101000667300 Homo sapiens WD repeat-containing protein 19 Proteins 0.000 description 1
- 241000701041 Human betaherpesvirus 7 Species 0.000 description 1
- 241000701027 Human herpesvirus 6 Species 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 101000666843 Orgyia pseudotsugata multicapsid polyhedrosis virus Uncharacterized 24.0 kDa protein Proteins 0.000 description 1
- 101000768120 Oryza nivara Uncharacterized protein ycf76 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102100039744 WD repeat-containing protein 19 Human genes 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000010460 detection of virus Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 229940124589 immunosuppressive drug Drugs 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 208000017805 post-transplant lymphoproliferative disease Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a primer probe composition for simultaneously detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) and application thereof. The primer composition disclosed by the invention does not interfere with each other, has better detection sensitivity and accuracy, is simple and convenient to operate, and can be widely applied to clinical diagnosis of EBV (Epstein-Barr Virus) and Human Cytomegalovirus (HCMV).
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a primer probe composition for simultaneously detecting EBV (Epstein-Barr Virus) and Human Cytomegalovirus (HCMV) and application thereof.
Background
Epstein-Barr Virus (EBV) is a gamma subfamily of the human herpesvirus, also known as human herpesvirus type 4; human Cytomegalovirus (HCMV) is a subfamily of the Human genus herpesvirus, also known as Human herpesvirus type 5. Both EBV and HCMV are double-stranded DNA viruses. More than 100 herpesviruses have been discovered so far, of which at least 8 are associated with human infections, including: HSV-1, HSV-2, Varicella Zoster Virus (VZV), EBV, cytomegalovirus (HCMV) and HHV-6, HHV-7, HHV-8.
In addition to the typical morphological characteristics of herpes viruses, the most important biological characteristics of 8 human herpes viruses are that the viruses persist in vivo after primary infection, so that the viruses are easily activated periodically in a herpes virus infected person, and the viruses are still infectious after being activated. Therefore, the herpes virus infected person can secrete one or more herpes viruses at any time, so that the common population keeps a higher infection rate. Of these, EBV and HCMV are the most harmful viruses, and EBV is known to be associated with African Burkitt's lymphoma, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease. HCMV can lead to congenital malformations and even dead fetuses in pregnant women after infection.
EBV and HCMV infections are common, and most people are infected with EB and HCMV in childhood, and are often acute or latent without clinical symptoms, and are immune due to the infection. In special cases, such as autoimmune deficiencies, acquired immune competence, administration of immunosuppressive drugs, etc., EB and HCMV viruses can easily activate infection. Most HCMV infections clinically manifest as EBV infections, and these 2 viruses are important pathogens of persistent febrile illnesses. In clinical tests, EBV and HCMV infection sometimes cannot be distinguished only by clinical manifestations, and it is necessary to detect both EBV and HCMV, and in addition, aids patients and organ transplant patients are also screened for EBV and HCMV.
Diagnosis of EBV and HCMV infection includes serological examinations, virus marker examinations, virus isolation. At present, serological marker detection is generally used as an auxiliary diagnosis method for acute or chronic EBV and HCMV infection, and anti-VCA-IgM and HCMV-IgM/IgG detection is an important index of acute primary infection. However, the serological markers actually detect the immune response state of the human body to infection, and cannot detect the virus, so that the infection cannot be accurately and sensitively reflected. In addition, with the development of technology, quantitative detection of viruses is becoming possible. For example, patent CN201410026784.5 discloses a real-time fluorescence quantitative PCR kit for simultaneously detecting common human herpesviruses HSV-1, HSV-2, EBV and HCMV, which consists of quantitative PCR reaction liquid, quantitative reaction liquid, HSV-1 standard substance, HSV-2 standard substance, EBV standard substance, HCMV standard substance, negative control substance, HSV-1 positive control substance, HSV-2 positive control substance, EBV positive control substance, HCMV positive control substance, instructions and a box body. The patent CN201710118846.9 discloses a reagent for rapidly detecting each subtype of human herpes virus, which comprises forward and reverse primers and corresponding internal reference samples for specifically amplifying HSV-1RL2, HSV-2UL28, VZV ORF26, EBV EBNA1, HCMV IE, HHV-6A or HHV-6B U22, HHV-6A or HHV-6B IE, HHV-7U95 and KSHV ORF72, can be used for rapidly and effectively identifying and diagnosing each subtype of early-stage infection clinical samples from different sources, and has high clinical diagnosis and use values.
At present, the relation between serological markers and EBV virus and HCMV virus copies is not established, so that a primer probe composition and a kit thereof which can directly judge the virus replication activity of EBV and HCMV viruses and have high detection accuracy and sensitivity are urgently needed to be provided.
Disclosure of Invention
In order to solve the above problems in the prior art, the present application provides a primer probe composition for simultaneously detecting EBV virus and human cytomegalovirus HCMV, and applications thereof. The primer composition disclosed by the invention does not interfere with each other, has better detection sensitivity and accuracy, is simple and convenient to operate, and can be widely applied to clinical diagnosis of EBV (Epstein-Barr Virus) and Human Cytomegalovirus (HCMV).
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the invention provides a primer probe composition for detecting EBV and HCMV, and the primer probe composition comprises an HCMV detection primer probe composition, an EBV detection primer probe composition and an internal standard detection primer probe composition.
Specifically, the genome of the HCMV virus is about 250kb and can encode a plurality of proteins, but the IE1 encoded by UL123 and the IE2 encoded by UL122 are the most expressed and important. IE1 is considered to be a transactivator for HCMV and host cell gene expression, and is the first protein to be synthesized following viral infection, and is essential for viral gene expression and replication in low MOI infections. IE2 is the major activator of the HCMV lytic cycle and has an irreplaceable effect on viral replication. As the most abundant protein expressed by HCMV, IE proteins not only play an important role in fighting against the innate and specific immunity of host cells, but also influence the latent and active state of the virus through a variety of mechanisms. Therefore, aiming at the IE1 gene, the conserved sequence of the gene is aligned by the GeneBank database of NCBI (national center for biological information) and a primer probe sequence is designed, wherein the target sequence of the primer probe sequence is the sequence shown in SEQ ID NO. 1.
Specifically, the total length of the EB virus genome is about 172 kb. The Bam H1W fragment is 3072bp long, highly conserved and repeated 12 times in genome, and the repeated sequence is flanked by short (us) and long (uL) unique sequence regions. Therefore, aiming at the Bam H1W gene, a great number of conserved sequences of the gene are aligned through a GeneBank database of NCBI (national center for biological information of the United states), and a primer probe sequence is designed, wherein the target sequence of the primer probe sequence is a sequence shown as SEQ ID NO. 5.
Specifically, the internal standard adopts a human RNaseP gene fragment, and the target sequence of the internal standard is the sequence shown in SEQ ID NO. 9.
Further specifically, the primer composition comprises:
(1) HCMV detection primer probe composition:
forward primer HCMVF: 5'-TCAGCATGTGCTCCTTGATTCT-3' (SEQ ID NO.2)
Reverse primer HCMVR: 5'-GTGTTGTTATCCTCCTCTACAGTCAAAC-3' (SEQ ID NO.3)
Probe HCMVP: 5'-TGCCGCACCATGTCCACTCGAA-3' (SEQ ID NO. 4);
(2) EBV detection primer probe composition:
forward primer EBF: 5'-AAGTGGTCCTGCAGCTATTTCTG-3' (SEQ ID NO.6)
Reverse primer EBR: 5'-AGAAGACCCCCTCTTACATTTGTG-3' (SEQ ID NO.7)
And (3) probe EBP: 5'-CGCATCAGAGCGCCAGGAGTCC-3' (SEQ ID NO. 8);
(3) internal standard detection primer probe composition:
forward primer ICF: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID NO.10)
Reverse primer ICR: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID NO.11)
Probe ICP: 5'-TTCTGACCTGAAGGCTCTGCGCG-3' (SEQ ID NO. 12).
Further concretely, the 5' end fluorescent label of the probe (SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.10) is selected from three different groups of FAM group, VIC/HEX/JOE group, ROX group, CY5 group, CY3 group and TAMRA group; the corresponding quenching group is selected from one or more of BHQ-1, BHQ-2 or BHQ-3.
Preferably, the HCMV fluorescent probe HCMVP (SEQ ID No.4) is labeled with FAM-BHQ-1; EBV fluorescent probe EBP (SEQ ID No.8) was labeled with VIC-BHQ-1; an internal standard fluorescent probe ICP (SEQ ID No.12) was labeled with CY 5-BHQ-2.
Alternatively, the ROX probe is used as a reference fluorescence for calibrating the difference in fluorescence signal between different reaction wells.
In another aspect, the invention provides an application of the primer probe composition in preparation of products for detecting EBV and HCMV viruses.
Specifically, the product includes but is not limited to a separate reagent, chip or kit.
In yet another aspect, the present invention provides a product for detecting EBV and HCMV viruses, wherein the product comprises the primer probe composition.
Specifically, the product includes but is not limited to a separate reagent, chip or kit.
Specifically, the product also comprises a PCR reaction solution.
More specifically, the PCR reaction solution includes 50mM tris (hydroxymethyl) aminomethane (pH6.8), 75mM potassium acetate, 3.0mM magnesium acetate, 0.05mM disodium EDTA, 5U Hot Start Taq DNA polymerase, 0.2U thermolabile UNG enzyme, 0.2mM dNTPs, 0.1% BSA, 0.02% Proclin300, 0.2. mu.M each of primers (HCMVF, HCMVR, EBF, EBR, ICF, ICR), and 0.1. mu.M each of fluorescent probes (HCMVP, EBP, ICP). Alternatively, 0.05. mu.M ROX probe was added as a reference fluorescence.
Specifically, the product further comprises a quality control product: negative control, EBV positive control, HCMV positive control, internal standard.
More specifically, the negative control is serum negative for EBV and HCMV DNA.
Further specifically, the HCMV positive control is a DNA pseudovirus containing an HCMV target sequence (SEQ ID NO. 1);
further specifically, the EBV positive control is a DNA pseudovirus containing an EBV target sequence (SEQ ID NO. 5);
more specifically, the internal standard is a cloned plasmid containing a human RNaseP gene fragment (SEQ ID NO. 9).
In particular, the product further comprises a rapid nucleic acid release agent for lysing cells and releasing viral nucleic acid.
More specifically, the rapid nucleic acid releasing agent comprises 100-200mM KOH or NaOH, 1-5% CA-630 or NP-40 or Twen-20, 5-10% LLS or SDS, and 100-200. mu.M Surfactin.
In another aspect, the invention provides the application of the primer probe composition or the product in detecting EBV and HCMV viruses.
In still another aspect, the present invention provides a method for detecting EBV and HCMV, which is a non-disease diagnosis and treatment method, comprising detecting EBV and HCMV in a test sample using the primer probe composition or the product.
Specifically, the method comprises the following steps:
(1) extracting DNA of a sample;
(2) amplifying the sample DNA extracted and purified in the step (1) by using the primer probe composition;
(3) and analyzing the amplification result.
Compared with the prior art, the kit provided by the invention has the following beneficial effects:
(1) the primer probe composition provided by the invention is used for simultaneously detecting the EBV virus and the human cytomegalovirus HCMV, has the advantages of good sensitivity and accuracy, high amplification efficiency, simple and convenient operation, quickness and time saving, and can be widely applied to clinical diagnosis of the EBV virus and the human cytomegalovirus HCMV.
(2) The primer probe composition (HCMV detection primer probe composition, EBV detection primer probe composition and internal standard detection primer probe composition) does not interfere with each other, and does not influence the accuracy and sensitivity of detection.
Drawings
FIG. 1 is a graph of FAM channel amplification in the detection of HCMV samples.
FIG. 2 is a graph of VIC channel amplification for the detection of EBV samples.
FIG. 3 is a CY5 channel amplification plot of IC as an internal standard of detection.
FIG. 4 is a graph of amplification profiles for all channels. .
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
The examples, where no specific techniques or conditions are indicated, are carried out according to the techniques or conditions described in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, ed. by Huang Pe, et al) or according to the instructions of the product.
Example 1 primer Probe composition for detecting EBV and HCMV viruses
The primer probe composition comprises an HCMV detection primer probe composition, an EBV detection primer probe composition and an internal standard detection primer probe composition. The primer probe is synthesized by the corporation of Shanghai Biotechnology engineering (Shanghai).
Specifically, the genome of the HCMV virus is about 250kb and can encode a plurality of proteins, but the IE1 encoded by UL123 and the IE2 encoded by UL122 are the most expressed and important. IE1 is considered to be a transactivator for HCMV and host cell gene expression, and is the first protein to be synthesized following viral infection, and is essential for viral gene expression and replication in low MOI infections. IE2 is the major activator of the HCMV lytic cycle and has an irreplaceable effect on viral replication. As the most abundant protein expressed by HCMV, IE proteins not only play an important role in fighting against the innate and specific immunity of host cells, but also influence the latent and active state of the virus through a variety of mechanisms. Therefore, aiming at the IE1 gene, the conserved sequence of the gene is aligned by the GeneBank database of NCBI (national center for biological information) and a primer probe sequence is designed, wherein the target sequence of the primer probe sequence is the sequence shown in SEQ ID NO. 1.
Specifically, the total length of the EB virus genome is about 172 kb. The Bam H1W fragment is 3072bp long, highly conserved and repeated 12 times in genome, and the repeated sequence is flanked by short (us) and long (uL) unique sequence regions. Therefore, aiming at the Bam H1W gene, a great number of conserved sequences of the gene are aligned through a GeneBank database of NCBI (national center for biological information of the United states), and a primer probe sequence is designed, wherein the target sequence of the primer probe sequence is a sequence shown as SEQ ID NO. 5.
Specifically, the internal standard adopts a human RNaseP gene fragment, and the target sequence of the internal standard is the sequence shown in SEQ ID NO. 9.
Further specifically, the primer composition comprises:
(1) HCMV detection primer probe composition:
forward primer HCMVF: 5'-TCAGCATGTGCTCCTTGATTCT-3' (SEQ ID NO.2)
Reverse primer HCMVR: 5'-GTGTTGTTATCCTCCTCTACAGTCAAAC-3' (SEQ ID NO.3)
Probe HCMVP: 5'-TGCCGCACCATGTCCACTCGAA-3' (SEQ ID NO. 4);
(2) EBV detection primer probe composition:
forward primer EBF: 5'-AAGTGGTCCTGCAGCTATTTCTG-3' (SEQ ID NO.6)
Reverse primer EBR: 5'-AGAAGACCCCCTCTTACATTTGTG-3' (SEQ ID NO.7)
And (3) probe EBP: 5'-CGCATCAGAGCGCCAGGAGTCC-3' (SEQ ID NO. 8);
(3) internal standard detection primer probe composition:
forward primer ICF: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID NO.10)
Reverse primer ICR: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID NO.11)
Probe ICP: 5'-TTCTGACCTGAAGGCTCTGCGCG-3' (SEQ ID NO. 12).
Further specifically, HCMV fluorescent probe HCMVP (SEQ ID No.4) was labeled with FAM-BHQ-1; EBV fluorescent probe EBP (SEQ ID No.8) was labeled with VIC-BHQ-1; an internal standard fluorescent probe ICP (SEQ ID No.12) was labeled with CY 5-BHQ-2.
Alternatively, the ROX probe is used as a reference fluorescence for calibrating the difference in fluorescence signal between different reaction wells.
Example 2 kit for detecting EBV and HCMV viruses
The compositions of the kit (24 test/kit) for EBV and HCMV viruses according to the present invention are shown in table 1 below.
TABLE 1
| Components | Loading capacity |
| PCR reaction solution | 960μL |
| Negative control | 50μL |
| EBV positive control | 50μL |
| HCMV positive control | 50μL |
| Internal standard | 50μL |
| Fast nucleic acid releasing agent | 120μL |
Specifically, the PCR reaction solution comprises 50mM tris (hydroxymethyl) aminomethane (pH6.8), 75mM potassium acetate, 3.0mM magnesium acetate, 0.05mM disodium EDTA, 5U hot-start Taq DNA polymerase, 0.2U thermolabile UNG enzyme, 0.2mM dNTPs, 0.1% BSA, 0.02% Proclin300, 0.2. mu.M each of primers (HCMVF, HCMVR, EBF, EBR, ICF, ICR), 0.1. mu.M each of fluorescent probes (HCMVP, EBP, ICP), and 0.05. mu.M ROX probe as reference fluorescence.
Specifically, the negative control is serum negative for EBV and HCMV DNA.
Specifically, the HCMV positive control is a DNA pseudovirus containing an HCMV target sequence (SEQ ID NO. 1).
Specifically, the EBV positive control is a DNA pseudovirus containing an EBV target sequence (SEQ ID NO. 5).
Specifically, the internal standard is a cloned plasmid containing a human RNaseP gene fragment (SEQ ID NO. 9).
The desired pseudovirus and internal standard cloning plasmid were synthesized by Xiamen-derived organisms or Biotechnology (Shanghai) Co., Ltd., and then 1X 104Pseudoviruses at copies/mL concentrations were formulated as positive controls and internal standards.
Specifically, the rapid nucleic acid release agent contains 100-200mM KOH or NaOH, 1-5% CA-630 or NP-40 or Twen-20, 5-10% LLS or SDS, and 100-200. mu.M Surfactin.
Example 3 method of Using the kit
(1) Sample treatment: and (3) adding 5 mu L of the rapid nucleic acid releasing agent into the PCR tube/plate, adding 5 mu L of the serum sample to be detected into the PCR tube filled with the rapid nucleic acid releasing agent, sucking, uniformly mixing, and standing at room temperature for 10 min.
(2) Preparing a reaction system: to the treated sample, 40. mu.L of the PCR reaction solution was added to complete the preparation of 50. mu.L of the amplification reaction system.
(3) And (3) PCR amplification: the in-silico amplification was performed according to the reaction program shown in Table 2 below.
TABLE 2
Detecting a fluorescence channel: HCMV DNA (FAM), EBV DNA (VIC), IC (CY5), ROX was chosen as reference fluorescence.
(4) Interpretation of results (as in table 3 below):
TABLE 3
Experimental example 1
A total of 88 serum samples isolated from peripheral blood were tested using the kit of example 2, with 24 HCMV-positive, 16 EBV-positive and 48-negative. Compared with the detection result of the reference kit, the kit detects 24 positive HCMV, 24/24 (100%) positive coincidence rate, 16 positive EBV, 16/16 (100%) positive coincidence rate, 48 negative coincidence rate and 48/48 (100%).
Meanwhile, 10 mixed samples of the HCMV positive sample and the EBV positive sample are taken, and the detection rate of the HCMV and EBV positive coincidence rate of the kit is 100 percent.
The experimental results show that the kit disclosed by the invention is simple to operate, convenient to process samples and good in detection accuracy, can be used for simultaneously detecting 2 viruses in a single tube, and saves the cost and time.
The above are merely embodiments of the present invention, which are described in detail and with particularity, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the spirit of the present invention, and these changes and modifications are within the scope of the present invention.
SEQUENCE LISTING
<110> Shandong microbial science and technology Limited
<120> primer probe composition for detecting EBV and HCMV viruses and application thereof
<130> 20220117
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 101
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
gggcatattt tttcagcatg tgctccttga ttctatgccg caccatgtcc actcgaacct 60
taatctgttt gactgtagag gaggataaca acacatataa g 101
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
tcagcatgtg ctccttgatt ct 22
<210> 3
<211> 28
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
gtgttgttat cctcctctac agtcaaac 28
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
tgccgcacca tgtccactcg aa 22
<210> 5
<211> 150
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
acgtaagcca gacagcagcc aattgtcagt tctagggagg gggaccactg cccctggtat 60
aaagtggtcc tgcagctatt tctggtcgca tcagagcgcc aggagtccac acaaatgtaa 120
gagggggtct tctacctctc cctagccctc 150
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
aagtggtcct gcagctattt ctg 23
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
agaagacccc ctcttacatt tgtg 24
<210> 8
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
cgcatcagag cgccaggagt cc 22
<210> 9
<211> 70
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
tcagatttgg acctgcgagc gggttctgac ctgaaggctc tgcgcggact tgtggagaca 60
gccgctcaag 70
<210> 10
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
agatttggac ctgcgagcg 19
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
ttctgacctg aaggctctgc gcg 23
Claims (10)
1. A primer probe composition for detecting EBV and HCMV viruses is characterized in that: the primer probe composition comprises an HCMV detection primer probe composition, an EBV detection primer probe composition and an internal standard detection primer probe composition;
(1) HCMV detection primer probe composition:
forward primer HCMVF: 5'-TCAGCATGTGCTCCTTGATTCT-3'
Reverse primer HCMVR: 5'-GTGTTGTTATCCTCCTCTACAGTCAAAC-3'
Probe HCMVP: 5'-TGCCGCACCATGTCCACTCGAA-3', respectively;
(2) EBV detection primer probe composition:
forward primer EBF: 5'-AAGTGGTCCTGCAGCTATTTCTG-3'
Reverse primer EBR: 5'-AGAAGACCCCCTCTTACATTTGTG-3'
And (3) probe EBP: 5'-CGCATCAGAGCGCCAGGAGTCC-3', respectively;
(3) internal standard detection primer probe composition:
forward primer ICF: 5'-AGATTTGGACCTGCGAGCG-3'
Reverse primer ICR: 5'-GAGCGGCTGTCTCCACAAGT-3'
Probe ICP: 5'-TTCTGACCTGAAGGCTCTGCGCG-3' are provided.
2. The primer probe composition of claim 1, wherein: the HCMV detection primer probe composition is used for detecting a sequence shown by SEQ ID NO.1, the EBV detection primer probe composition is used for detecting a sequence shown by SEQ ID NO.5, and the internal standard detection primer probe composition is used for detecting a sequence shown by SEQ ID NO. 9.
3. The primer probe composition of claim 2, wherein: the 5' end fluorescent label of the probe is selected from three different FAM groups, VIC/HEX/JOE groups, ROX groups, CY5 groups, CY3 groups and TAMRA groups; the quenching group is selected from one or more of BHQ-1, BHQ-2 or BHQ-3.
4. The primer probe composition of claim 3, wherein: the probe HCMVP is marked by FAM-BHQ-1; the probe EBP is marked by VIC-BHQ-1; the probe ICP was labeled with CY 5-BHQ-2.
5. Use of the primer probe composition according to any one of claims 1 to 4 for the preparation of a product for the detection of EBV and HCMV viruses.
6. A product for detecting EBV and HCMV viruses, comprising: the product comprises the primer probe composition of any one of claims 1 to 4.
7. The product of claim 6, wherein: the product also comprises PCR reaction solution, and quality control products: negative control, EBV positive control, HCMV positive control, internal standard, and rapid nucleic acid release agent.
8. The product of claim 7, wherein: the PCR reaction solution comprises 50mM of tris (hydroxymethyl) aminomethane (pH6.8), 75mM of potassium acetate, 3.0mM of magnesium acetate, 0.05mM of disodium EDTA, 5U of hot start Taq DNA polymerase, 0.2U of thermolabile UNG enzyme, 0.2mM of dNTPs, 0.1% of BSA, 0.02% of Proclin300, 0.2 mu M of each primer and 0.1 mu M of each fluorescent probe.
9. The product of claim 8, wherein: the negative control is serum negative to EBV and HCMV DNA; the HCMV positive control is a DNA pseudovirus containing an HCMV target sequence SEQ ID NO. 1; the EBV positive control is a DNA pseudovirus containing an EBV target sequence SEQ ID NO. 5; the internal standard is a clone plasmid containing a human RNaseP gene fragment SEQ ID NO. 9.
10. The product of claim 9, wherein: the rapid nucleic acid releasing agent contains 100-.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210118685.4A CN114350858A (en) | 2022-02-08 | 2022-02-08 | Primer probe composition for detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) viruses and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210118685.4A CN114350858A (en) | 2022-02-08 | 2022-02-08 | Primer probe composition for detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) viruses and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114350858A true CN114350858A (en) | 2022-04-15 |
Family
ID=81092608
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210118685.4A Pending CN114350858A (en) | 2022-02-08 | 2022-02-08 | Primer probe composition for detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) viruses and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114350858A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122396A (en) * | 2013-01-10 | 2013-05-29 | 湖南圣湘生物科技有限公司 | Kit for detecting human cytomegalovirus (HCMV) |
| CN103642945A (en) * | 2013-12-30 | 2014-03-19 | 上海星耀医学科技发展有限公司 | Reference-containing high-sensitivity fluorescent quantitative polymerase chain reaction (PCR) kit for Epstein-Barr virus |
| CN110241256A (en) * | 2019-05-27 | 2019-09-17 | 江苏达伯药业有限公司 | A kind of kit of multiple quantitative detection Epstein-Barr virus and cytomegalovirus |
| CN111286560A (en) * | 2020-03-19 | 2020-06-16 | 申联生物医药（上海）股份有限公司 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
-
2022
- 2022-02-08 CN CN202210118685.4A patent/CN114350858A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122396A (en) * | 2013-01-10 | 2013-05-29 | 湖南圣湘生物科技有限公司 | Kit for detecting human cytomegalovirus (HCMV) |
| CN103642945A (en) * | 2013-12-30 | 2014-03-19 | 上海星耀医学科技发展有限公司 | Reference-containing high-sensitivity fluorescent quantitative polymerase chain reaction (PCR) kit for Epstein-Barr virus |
| CN110241256A (en) * | 2019-05-27 | 2019-09-17 | 江苏达伯药业有限公司 | A kind of kit of multiple quantitative detection Epstein-Barr virus and cytomegalovirus |
| CN111286560A (en) * | 2020-03-19 | 2020-06-16 | 申联生物医药（上海）股份有限公司 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
Non-Patent Citations (4)
| Title |
|---|
| 姜静等: "《分子生物学实验原理与技术》", 31 March 2003, 东北林业大学出版社 * |
| 曹莉莉等: "人巨细胞病毒的PCR检测", 《青岛大学医学院学报》 * |
| 沈佐君: "《分子诊断标准化操作程序》", 31 October 2019, 上海科学技术出版社 * |
| 马艳萍等: "荧光定量聚合酶链式反应法检测婴儿尿液人巨细胞病毒DNA对人巨细胞病毒感染的诊断意义", 《实用儿科临床杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kimura et al. | Measuring Epstein–Barr virus (EBV) load: the significance and application for each EBV‐associated disease | |
| EP2007910B1 (en) | Method and microarray for detecting herpesviruses | |
| Achour et al. | Human herpesvirus-6 (HHV-6) DNA in plasma reflects the presence of infected blood cells rather than circulating viral particles | |
| CN103820573A (en) | Fluorescent quantitation PCR (polymerase chain reaction) detection kit for four conventional herpesvirus hominises | |
| CN111088401A (en) | Multi-virus detection primer and kit thereof | |
| CN103773898A (en) | Triple detection kit for human herpes viruses HSV-1, HSV-2 and HCMV | |
| CN111500788A (en) | Kit for detecting human herpesvirus infection and detection method thereof | |
| CN111304363A (en) | EB virus nucleic acid quantitative determination kit | |
| EP2663658B1 (en) | Materials and method for detecting cytomegalovirus (cmv) | |
| Gao et al. | Rapid internal control reference recombinase-aided amplification assays for EBV and CMV detection | |
| CN103509880A (en) | LAMP detection kit of highly-pathogenic porcine reproductive and respiratory syndrome viruses | |
| Kuhara et al. | Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification | |
| CN105779644A (en) | Realtime fluorescent nucleic acid constant temperature amplification detection kit of human cytomegalovirus (HCMV) | |
| Deback et al. | Detection of human herpesviruses HHV-6, HHV-7 and HHV-8 in whole blood by real-time PCR using the new CMV, HHV-6, 7, 8 R-gene™ kit | |
| CN110846439A (en) | HCMV detection product and application thereof | |
| CN103820574A (en) | Real-time fluorescent quantitation PCR (polymerase chain reaction) parting detection kit for human herpesvirus-6 | |
| CN114350858A (en) | Primer probe composition for detecting EBV (Epstein-Barr Virus) and HCMV (human cytomegalovirus) viruses and application thereof | |
| CN115873993A (en) | Kit for detecting 9 genotypes of hepatitis B virus and application thereof | |
| CN116144841A (en) | Primer and probe combination for detecting bovine polyoma virus and application thereof | |
| CN114703320A (en) | Primer, probe and kit for rapid screening and quantitative detection of multiple viruses before and after organ transplantation | |
| CN114540538A (en) | Detection kit for simultaneously and quantitatively detecting EHV-1 and EHV-4 and application thereof | |
| Cardoso et al. | Phylogenetic analysis of the DNA polymerase gene of a novel alphaherpesvirus isolated from an Indian Gyps vulture | |
| CN107653345B (en) | Real-time fluorescence quantitative PCR method for simian B virus, universal primer, MGB probe and kit thereof | |
| KR101099041B1 (en) | Multiplex PCR primers and detection method for diagnosing herpesviruses | |
| WO2016071925A2 (en) | TITLE: INTEGRATION OF β-ACTIN GENE FOR SAMPLE QUALITY CHECK IN HSV-1 AND HSV-2 DIAGNOSTIC KIT |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220415 |
|
| RJ01 | Rejection of invention patent application after publication |