CN106048088B - LAMP primer combination for detecting 4 ophthalmic infectious viruses and application - Google Patents
LAMP primer combination for detecting 4 ophthalmic infectious viruses and application Download PDFInfo
- Publication number
- CN106048088B CN106048088B CN201610404661.XA CN201610404661A CN106048088B CN 106048088 B CN106048088 B CN 106048088B CN 201610404661 A CN201610404661 A CN 201610404661A CN 106048088 B CN106048088 B CN 106048088B
- Authority
- CN
- China
- Prior art keywords
- primer
- sequence
- virus
- herpes simplex
- simplex virus
- 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.)
- Active
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 69
- 208000015181 infectious disease Diseases 0.000 title abstract description 16
- 230000002458 infectious effect Effects 0.000 title abstract description 8
- 108020004414 DNA Proteins 0.000 claims abstract description 149
- 241000700584 Simplexvirus Species 0.000 claims abstract description 85
- 241000701085 Human alphaherpesvirus 3 Species 0.000 claims abstract description 71
- 241000701022 Cytomegalovirus Species 0.000 claims abstract description 65
- 102000053602 DNA Human genes 0.000 claims abstract description 61
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 49
- 241000701074 Human alphaherpesvirus 2 Species 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 22
- 230000003321 amplification Effects 0.000 claims description 94
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 94
- 238000006243 chemical reaction Methods 0.000 claims description 36
- 102100031822 Optineurin Human genes 0.000 claims description 14
- 101710131459 Optineurin Proteins 0.000 claims description 14
- 101000686153 Homo sapiens Ras-related GTP-binding protein A Proteins 0.000 claims description 10
- 102100025001 Ras-related GTP-binding protein A Human genes 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 238000002405 diagnostic procedure Methods 0.000 claims 2
- 239000000523 sample Substances 0.000 description 61
- 239000013612 plasmid Substances 0.000 description 41
- 238000007397 LAMP assay Methods 0.000 description 38
- 230000006870 function Effects 0.000 description 24
- 239000002773 nucleotide Substances 0.000 description 24
- 125000003729 nucleotide group Chemical group 0.000 description 24
- 239000000203 mixture Substances 0.000 description 20
- 238000001514 detection method Methods 0.000 description 14
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 9
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 8
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 8
- 229960003237 betaine Drugs 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 102100022219 NF-kappa-B essential modulator Human genes 0.000 description 6
- 101710090077 NF-kappa-B essential modulator Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 208000005100 Herpetic Keratitis Diseases 0.000 description 4
- 206010073938 Ophthalmic herpes simplex Diseases 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 201000006082 Chickenpox Diseases 0.000 description 3
- 208000007514 Herpes zoster Diseases 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 206010046980 Varicella Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 201000004569 Blindness Diseases 0.000 description 2
- 206010048843 Cytomegalovirus chorioretinitis Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 208000001763 cytomegalovirus retinitis Diseases 0.000 description 2
- 230000010460 detection of virus Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 208000030533 eye disease Diseases 0.000 description 2
- 210000004392 genitalia Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 208000004142 Acute Retinal Necrosis Syndrome Diseases 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 206010061788 Corneal infection Diseases 0.000 description 1
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 206010015958 Eye pain Diseases 0.000 description 1
- 206010060919 Foetal malformation Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 208000029523 Interstitial Lung disease Diseases 0.000 description 1
- 206010022941 Iridocyclitis Diseases 0.000 description 1
- 206010065119 Necrotising herpetic retinopathy Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010061323 Optic neuropathy Diseases 0.000 description 1
- 201000010183 Papilledema Diseases 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 201000007737 Retinal degeneration Diseases 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- 206010038886 Retinal oedema Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000005181 Varicella Zoster Virus Infection Diseases 0.000 description 1
- 206010053648 Vascular occlusion Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 201000004612 anterior uveitis Diseases 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 201000004709 chorioretinitis Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 206010014801 endophthalmitis Diseases 0.000 description 1
- 210000004920 epithelial cell of skin Anatomy 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 230000007160 gastrointestinal dysfunction Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 206010023365 keratopathy Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000005906 menstruation Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 208000020911 optic nerve disease Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 201000011195 retinal edema Diseases 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 231100000046 skin rash Toxicity 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 208000002254 stillbirth Diseases 0.000 description 1
- 231100000537 stillbirth Toxicity 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 208000010531 varicella zoster infection Diseases 0.000 description 1
- 230000006496 vascular abnormality Effects 0.000 description 1
- 208000021331 vascular occlusion disease Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000004382 visual function Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a LAMP primer combination for detecting 4 ophthalmic infectious viruses and application thereof. The primer combination of the invention consists of 24 single-stranded DNA molecules shown in sequences 1 to 24. The invention also protects the application of the primer combination. The invention also discloses a method for identifying whether the virus to be detected is herpes simplex virus I type, herpes simplex virus II type, varicella-zoster virus or cytomegalovirus, a method for identifying whether the virus to be detected is herpes simplex virus I type or herpes simplex virus II type or varicella-zoster virus or cytomegalovirus, and a method for identifying whether the sample to be detected is infected with the herpes simplex virus I type and/or the herpes simplex virus II type and/or the varicella-zoster virus and/or the cytomegalovirus. By applying the LAMP primer and the method, the herpes simplex virus I, the herpes simplex virus II, the varicella-zoster virus and the cytomegalovirus can be quickly and accurately detected.
Description
Technical Field
The invention relates to a LAMP primer combination for detecting 4 ophthalmic infectious viruses and application thereof.
background
Herpes Simplex Virus (HSV) is the causative agent of viral herpes and is currently classified by antigenic differences into herpes simplex virus type I and herpes simplex virus type II. Type I is primarily responsible for infections of the skin, mucous membranes (oral mucosa) and organs (brain) outside the genitals. Type II primarily causes infections of the skin mucosa at genital sites. More than 50% of healthy people are carriers of the virus. HSV does not generate long-term immunity in human bodies, and when the body commensuration force is reduced, such as fever, gastrointestinal dysfunction, menstruation, pregnancy, focus infection and divination of motivation, hidden HSV in the bodies is activated to cause diseases, thereby affecting the health of human beings. The corneal infection caused by herpes simplex virus is called Herpes Simplex Keratitis (HSK), which is one of serious infectious eye diseases in the world today, and the incidence rate of the herpes simplex virus accounts for the first place of keratopathy. HSK blindness accounts for 42.8% of corneal blindness in China, and is the top. HSK has high morbidity, is difficult to cure and easy to relapse, seriously influences the visual function of a patient, and becomes one of the hot problems which are very concerned by the ophthalmology community at home and abroad.
cytomegalovirus (CMV), also known as cytomegavirus, is a virus of cytoinclusion bodies, and has a very wide infection in people, the infection rate of adults in china is more than 95%, usually recessive infection occurs, most infected people have no clinical symptoms, but serious diseases can be caused by attacking a plurality of organs and systems under certain conditions. The virus can invade lung, liver, kidney, salivary gland, mammary gland and other glands, and multinuclear leucocyte and lymphocyte, and can be discharged from saliva, milk sweat blood, urine, semen, uterus secretion for a long time or intermittently. In general, multiple routes of transmission, such as oral cavity, genital tract, placenta, blood transfusion or organ transplantation, have the potential of causing cancer. CMV retinitis is one of the major manifestations of CMV infection, and patients with CMV retinitis will develop symptoms and complications such as retinal edema, retinal vascular abnormalities (vascular occlusion), retinal detachment, chorioretinitis, etc., which, if untreated, will develop slowly and irreversibly, leaving paralysis-marks, blind spots, and retinal atrophy. CMV also causes optic neuropathy, which reduces vision rapidly.
Varicella-zoster virus (VZV), which causes chickenpox in childhood infections, remains latent in the body after recovery, and recurs to herpes zoster in adults in a small number of patients, is known as varicella-zoster virus. Humans are the only natural host for varicella-zoster virus, and skin epithelial cells are the major target cells. The vesicular skin rash contains a large amount of viruses, and the chickenpox is disappeared without leaving scars, so the disease condition is light, but the patients are occasionally complicated with interstitial pneumonia and encephalitis after infection. The chicken pox of children suffering from cellular immunodeficiency, leukemia, kidney diseases or long-term use of corticosteroids and antimetabolites is serious and even life-threatening. When adults suffer from varicella, 20-30% of concurrent pneumonia generally causes serious illness and high fatality rate. The pregnant women also have severe varicella manifestations and can cause fetal malformation, miscarriage or stillbirth. Varicella-zoster virus infection can cause zoster ophthalmia. Varicella-zoster virus can cause two clinical forms in addition to the traditional eye disease of herpes zoster: the first is acute retinal necrosis syndrome, which is manifested by the necrosis of the grey-white turbid retina widely around the bilateral retina, often with ocular pain accompanied by keratitis or iridocyclitis; another is progressive outer retinal necrosis syndrome, where the vitreous or anterior segment inflammatory response is not evident, but there may be macular foveal lesions early in the course of the disease.
The current clinical detection method aiming at the virus mainly comprises separation culture and serological diagnosis. The detection methods are complicated, long in detection time, low in sensitivity, easy to miss detection and error detection and incapable of meeting the requirement of rapid detection. The application of the molecular detection technology developed in recent years, particularly the PCR technology, in the aspects of quick identification and detection of microorganisms opens up a new way for quick detection of viruses. However, PCR has disadvantages of long detection time, susceptibility to contamination, and high false positive rate, and thus its application is limited. Loop-mediated isothermal amplification (LAMP) is a sensitive, specific, simple and rapid nucleic acid amplification technology developed in recent years, and the principle is that under the action of DNA polymerase with strand displacement activity, 4-6 primers of 6-8 regions are identified, a target gene is rapidly and specifically amplified under an isothermal condition, and the LAMP can be popularized and applied to rapid and accurate detection of viruses.
Disclosure of Invention
The invention aims to provide an LAMP primer combination for detecting 4 ophthalmic infectious viruses and application thereof.
The invention provides a primer combination, which consists of a primer group I, a primer group II, a primer group III and a primer group IV;
The primer group I consists of a primer F3-1, a primer B3-1, a primer FIP-1, a primer BIP-1, a primer LB-1 and a primer LF-1;
The primer F3-1 is (a1) or (a 2):
(a1) A single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the primer B3-1 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the primer FIP-1 is (a5) or (a6) as follows:
(a5) A single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a6) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;
the primer BIP-1 is (a7) or (a 8):
(a7) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a8) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
The primer LB-1 is (a9) or (a10) as follows:
(a9) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
(a10) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;
the primer LF-1 is (a11) or (a12) as follows:
(a11) A single-stranded DNA molecule shown in sequence 6 of the sequence table;
(a12) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same functions as the sequence 6;
The primer group II consists of a primer F3-2, a primer B3-2, a primer FIP-2, a primer BIP-2, a primer LB-2 and a primer LF-2;
the primer F3-2 is (b1) or (b 2):
(b1) a single-stranded DNA molecule shown in sequence 7 of the sequence table;
(b2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and having the same functions as the sequence 7;
the primer B3-2 is (B3) or (B4) as follows:
(b3) a single-stranded DNA molecule shown in sequence 8 of the sequence table;
(b4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 8 and have the same functions as the sequence 8;
The primer FIP-2 is (b5) or (b6) as follows:
(b5) a single-stranded DNA molecule shown in sequence 9 of the sequence table;
(b6) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 9 and has the same function as the sequence 9;
the primer BIP-2 is (b7) or (b8) as follows:
(b7) a single-stranded DNA molecule shown in sequence 10 of the sequence table;
(b8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 10 and has the same function as the sequence 10;
the primer LB-2 is (b9) or (b10) as follows:
(b9) a single-stranded DNA molecule shown in sequence 11 of the sequence table;
(b10) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 11 and having the same functions as the sequence 11;
The primer LF-3 is (b11) or (b12) as follows:
(b11) A single-stranded DNA molecule shown in sequence 12 of the sequence table;
(b12) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 12 and has the same function as the sequence 12;
The primer group III consists of a primer F3-3, a primer B3-3, a primer FIP-2, a primer BIP-3, a primer LB-3 and a primer LF-3;
The primer F3-3 is (c1) or (c2) as follows:
(c1) a single-stranded DNA molecule shown in sequence 13 of the sequence table;
(c2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 13 and has the same function as the sequence 13;
the primer B3-3 is (c3) or (c4) as follows:
(c3) a single-stranded DNA molecule shown as a sequence 14 in a sequence table;
(c4) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 14 and has the same function as the sequence 14;
the primer FIP-2 is (c5) or (c6) as follows:
(c5) A single-stranded DNA molecule shown in sequence 15 of the sequence table;
(c6) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 15 and having the same functions as the sequence 15;
the primer BIP-3 is (c7) or (c8) as follows:
(c7) a single-stranded DNA molecule shown as sequence 16 in the sequence table;
(c8) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 16 and having the same functions as the sequence 16;
The primer LB-3 is (c9) or (c10) as follows:
(c9) a single-stranded DNA molecule shown in sequence 17 of the sequence table;
(c10) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 17 and having the same functions as the sequence 17;
the primer LF-3 is (c11) or (c12) as follows:
(c11) A single-stranded DNA molecule shown in sequence 18 of the sequence table;
(c12) And (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 18 and has the same function as the sequence 18.
The primer group IV consists of a primer F3-4, a primer B3-4, a primer FIP-4, a primer BIP-4, a primer LB-4 and a primer LF-4;
The primer F3-4 is (d1) or (d 2):
(d1) a single-stranded DNA molecule shown as sequence 19 in the sequence table;
(d2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 19 and having the same functions as the sequence 19;
The primer B3-4 is (d3) or (d 4):
(d3) A single-stranded DNA molecule shown in sequence 20 of the sequence table;
(d4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 20 and having the same functions as the sequence 20;
the primer FIP-4 is (d5) or (d6) as follows:
(d5) A single-stranded DNA molecule shown in sequence 21 of the sequence table;
(d6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 21 and has the same function as the sequence 21;
The primer BIP-4 is (d7) or (d8) as follows:
(d7) a single-stranded DNA molecule shown as a sequence 22 in a sequence table;
(d8) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 22 and having the same functions as the sequence 22;
the primer LB-4 is (d9) or (d10) as follows:
(d9) a single-stranded DNA molecule shown as sequence 23 in the sequence table;
(d10) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 23 and having the same functions as the sequence 23;
The primer LF-4 is (d11) or (d12) as follows:
(d11) A single-stranded DNA molecule shown in sequence 24 of the sequence table;
(d12) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 24 and has the same function as the sequence 24.
the primer combination is used in any one of the following (e1) to (e 6):
(e1) identifying herpes simplex virus type I, herpes simplex virus type II, varicella-zoster virus and cytomegalovirus;
(e2) preparing a kit for identifying herpes simplex virus I, herpes simplex virus II, varicella-zoster virus and cytomegalovirus;
(e3) identifying whether the virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus;
(e4) preparing a kit for identifying whether the virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus;
(e5) identifying whether the sample to be detected is infected by herpes simplex virus I and/or herpes simplex virus II and/or varicella-zoster virus and/or cytomegalovirus;
(e6) preparing a kit for identifying whether a sample to be detected is infected by herpes simplex virus I and/or herpes simplex virus II and/or varicella-zoster virus and/or cytomegalovirus.
The invention also protects the application of the primer combination, which is any one of the following (e1) to (e 6):
(e1) identifying herpes simplex virus type I, herpes simplex virus type II, varicella-zoster virus and cytomegalovirus;
(e2) preparing a kit for identifying herpes simplex virus I, herpes simplex virus II, varicella-zoster virus and cytomegalovirus;
(e3) identifying whether the virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus;
(e4) Preparing a kit for identifying whether the virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus;
(e5) Identifying whether the sample to be detected is infected by herpes simplex virus I and/or herpes simplex virus II and/or varicella-zoster virus and/or cytomegalovirus;
(e6) preparing a kit for identifying whether a sample to be detected is infected by herpes simplex virus I and/or herpes simplex virus II and/or varicella-zoster virus and/or cytomegalovirus.
the invention also protects a kit containing the primer combination; the use of the kit is as follows (f1), (f2) or (f 3):
(f1) Identifying herpes simplex virus type I, herpes simplex virus type II, varicella-zoster virus and cytomegalovirus;
(f2) identifying whether the virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus;
(f3) identifying whether the sample to be tested is infected by herpes simplex virus I and/or herpes simplex virus II and/or varicella-zoster virus and/or cytomegalovirus.
the invention also provides a preparation method of the kit, which comprises the step of packaging each primer independently.
the invention also discloses a method for identifying whether the virus to be detected is herpes simplex virus I, herpes simplex virus II, varicella-zoster virus or cytomegalovirus, which comprises the following steps:
(1) Extracting the genome DNA of the virus to be detected;
(2) performing LAMP amplification reaction by respectively adopting the primer group I, the primer group II, the primer group III and the primer group IV of the primer combination by taking the genomic DNA obtained in the step (1) as a template, wherein positive amplification by taking the genomic DNA as the template and the virus to be detected as herpes simplex virus I can be realized by adopting the primer group I, positive amplification by taking the genomic DNA as the template and the virus to be detected as herpes simplex virus II can be realized by adopting the primer group II, positive amplification by taking the genomic DNA as the template and the virus to be detected as varicella-zoster virus can be realized by adopting the primer group III, and positive amplification by taking the genomic DNA as the template and the virus to be detected as cytomegalovirus can be realized by adopting the primer group IV.
in the method, the virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus.
the invention also discloses a method for identifying or assisting in identifying whether the virus to be detected is herpes simplex virus I type or herpes simplex virus II type or varicella-zoster virus or cytomegalovirus, which comprises the following steps:
(1) extracting the genome DNA of the virus to be detected;
(2) performing LAMP amplification reaction by respectively adopting the primer group I, the primer group II, the primer group III and the primer group IV of the primer combination by taking the genomic DNA obtained in the step (1) as a template, if the primer group I is adopted, realizing positive amplification by taking the genomic DNA as the template, and a virus to be detected or a candidate of the virus to be detected as a herpes simplex virus I type, if the primer group II is adopted, realizing positive amplification by taking the genomic DNA as the template, and a virus to be detected or a candidate of the virus to be detected as a herpes simplex virus II type, if the primer group III is adopted, realizing positive amplification by taking the genomic DNA as the template, and a virus to be detected as a varicella zoster virus, if the primer group IV is adopted, realizing positive amplification by taking the genomic DNA as the template, and a virus to be detected as a cytomegalovirus, and if the primer group I, the primer group II, the primer group III and the primer group IV are not adopted, can realize positive amplification by taking the genomic DNA as the template, The viruses to be tested are non-herpes simplex virus type I and non-herpes simplex virus type II and non-varicella-zoster virus and non-cytomegalovirus.
The invention also provides a method for identifying whether a sample to be detected is infected with herpes simplex virus I and/or herpes simplex virus II and/or varicella-zoster virus and/or cytomegalovirus, which comprises the following steps:
(1) Extracting the total DNA of a sample to be detected;
(2) performing LAMP amplification reaction by respectively adopting the primer group I, the primer group II, the primer group III and the primer group IV of the primer combination by taking the total DNA obtained in the step (1) as a template, if the primer group I is adopted, positive amplification by taking the total DNA as the template can be realized, a sample to be detected is suspected to be infected with the herpes simplex virus I type, if the primer group II is adopted, positive amplification by taking the total DNA as the template can be realized, the sample to be detected is suspected to be infected with the herpes simplex virus II type, if the primer group III is adopted, positive amplification by taking the total DNA as the template can be realized, the sample to be detected is suspected to be infected with the varicella-zoster virus, if the primer group IV is adopted, positive amplification by taking the total DNA as the template can be realized, the sample to be infected with the cytomegalovirus, and if the primer group I, the primer group II, the primer group III and the primer group IV are all adopted, the positive amplification by taking the, The sample to be tested is suspected to be not infected by herpes simplex virus I, is suspected to be not infected by herpes simplex virus II, is suspected to be not infected by varicella-zoster virus and is suspected to be not infected by cytomegalovirus.
in any of the above methods, the "positive amplification" can be specifically determined by the following method: detecting the fluorescence signal by a fluorescence PCR instrument, and if a positive amplification curve appears, carrying out positive amplification. The positive amplification curve may specifically be an S-type amplification curve.
the invention also protects the primer group I.
the application of the primer group I is (g1) or (g 2):
(g1) identifying whether the virus to be detected is herpes simplex virus I;
(g2) And identifying whether the sample to be detected is infected with the herpes simplex virus I.
The invention also protects the application of the primer group I in the preparation of a kit A, and the kit A is used as the following (g1) or (g 2):
(g1) identifying whether the virus to be detected is herpes simplex virus I;
(g2) And identifying whether the sample to be detected is infected with the herpes simplex virus I.
the invention also protects a kit A containing the primer group I, and the application of the kit A is as follows (g1) or (g 2):
(g1) identifying whether the virus to be detected is herpes simplex virus I;
(g2) and identifying whether the sample to be detected is infected with the herpes simplex virus I.
The invention also protects the primer group II.
the application of the primer group II is (g3) or (g 4):
(g3) identifying whether the virus to be detected is herpes simplex virus II;
(g4) and identifying whether the sample to be detected is infected with the herpes simplex virus II.
the invention also protects the application of the primer group II in the preparation of a kit B, and the application of the kit B is as follows (g3) or (g 4):
(g3) identifying whether the virus to be detected is herpes simplex virus II;
(g4) and identifying whether the sample to be detected is infected with the herpes simplex virus II.
the invention also protects a kit B containing the primer group II, and the application of the kit B is as follows (g3) or (g 4):
(g3) identifying whether the virus to be detected is herpes simplex virus II;
(g4) and identifying whether the sample to be detected is infected with the herpes simplex virus II.
the invention also protects the primer group III.
The application of the primer group III is (g5) or (g 6):
(g5) identifying whether the virus to be detected is varicella-zoster virus;
(g6) And identifying whether the sample to be detected is infected with varicella-zoster virus.
The invention also protects the application of the primer group III in the preparation of a kit C, and the application of the kit C is as follows (g5) or (g 6):
(g5) identifying whether the virus to be detected is varicella-zoster virus;
(g6) And identifying whether the sample to be detected is infected with varicella-zoster virus.
the invention also protects a kit C containing the primer group III, and the application of the kit C is as follows (g5) or (g 6):
(g5) identifying whether the virus to be detected is varicella-zoster virus;
(g6) and identifying whether the sample to be detected is infected with varicella-zoster virus.
the invention also protects the primer group IV.
the application of the primer group IV is (g7) or (g 8):
(g7) Identifying whether the virus to be detected is cytomegalovirus;
(g8) And identifying whether the sample to be detected is infected with the cytomegalovirus.
The invention also protects the application of the primer group IV in the preparation of a kit D, and the application of the kit D is as follows (g7) or (g 8):
(g7) identifying whether the virus to be detected is cytomegalovirus;
(g8) and identifying whether the sample to be detected is infected with the cytomegalovirus.
the invention also protects a kit D containing the primer group IV, and the application of the kit D is as follows (g7) or (g 8):
(g7) identifying whether the virus to be detected is cytomegalovirus;
(g8) and identifying whether the sample to be detected is infected with the cytomegalovirus.
any one of the above samples to be tested may be an eye clinical sample culture, an eye liquid or an aspirate.
in any one of the above reaction systems for LAMP amplification by using the primer group I, the concentration of the primer mixture in the primer group I is as follows: 0.5. mu. M F3-1, 0.5. mu.MB 3-1, 2. mu.M FIP-1, 2. mu.M BIP-1, 1. mu.M LF-1, 1. mu.M LB-1.
in any one of the above reaction systems for LAMP amplification by using the primer group II, the concentration of the primer mixture in the primer group II is as follows: 0.5. mu. M F3-2, 0.5. mu.MB 3-2, 2. mu.M FIP-2, 2. mu.M BIP-2, 1. mu.M LF-2, 1. mu.M LB-2.
in any one of the above reaction systems for LAMP amplification by using the primer group III, the concentration of the primer mixture in the primer group III is as follows: 0.5. mu. M F3-3, 0.5. mu.MB 3-3, 2. mu.M FIP-3, 2. mu.M BIP-3, 1. mu.M LF-3, 1. mu.M LB-3.
in any one of the above reaction systems for LAMP amplification by using the primer set IV, the concentration of the primer mixture in the primer set IV is as follows: 0.5. mu. M F3-4, 0.5. mu.MB 3-4, 2. mu.M FIP-4, 2. mu.M BIP-4, 1. mu.M LF-4, 1. mu.M LB-4.
Any one of the above reaction systems for LAMP amplification by using the primer group I may specifically be: 1 μ L of 10 XThermoPol Buffer, 1.6 μ L of 5M betaine, 0.1 μ L of 50mg/ml BSA, 0.4 μ L of 100mM MgSO40.3. mu.L of 20 × Eva Green, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (F3-1, B3-1, FIP-1, BIP-1, LF-1 and LB-1)Mixture), 50pg-50ng template DNA, using ddH2Make up to 10. mu.L of O.
any one of the above reaction systems for LAMP amplification by using the primer group II may specifically be: 1 μ L of 10 XThermoPol Buffer, 1.6 μ L of 5M betaine, 0.1 μ L of 50mg/ml BSA, 0.4 μ L of 100mM MgSO40.3. mu.L of 20 × EvaGreen, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-2, B3-2, FIP-2, BIP-2, LF-2 and LB-2), 50pg-50ng template DNA with ddH2Make up to 10. mu.L of O.
Any one of the above reaction systems for LAMP amplification by using the primer group III may specifically be: 1 μ L of 10 XThermoPol Buffer, 1.6 μ L of 5M betaine, 0.1 μ L of 50mg/ml BSA, 0.4 μ L of 100mM MgSO40.3. mu.L of 20 × EvaGreen, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-3, B3-3, FIP-3, BIP-3, LF-3 and LB-3), 50pg to 50ng template DNA with ddH2Make up to 10. mu.L of O.
any one of the above reaction systems for LAMP amplification by using the primer group IV may specifically be: 1 μ L of 10 XThermoPol Buffer, 1.6 μ L of 5M betaine, 0.1 μ L of 50mg/ml BSA, 0.4 μ L of 100mM MgSO40.3. mu.L of 20 × Eva Green, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-4, B3-4, FIP-4, BIP-4, LF-4 and LB-4), 50pg-50ng template DNA, made up to 10. mu.L with ddH 2O.
the reaction procedure of any one of the above LAMP amplifications may specifically be: keeping the temperature at 65 ℃ for 50 min. In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
The invention provides LAMP primers and a method for detecting 4 ophthalmic infectious viruses. The 4 viruses include: herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus and cytomegalovirus. The LAMP primers are 4 sets, each virus is 1 set, each set comprises outer primers F3 and B3, inner primers FIP and BIP and loop primers LF and LB, each set can specifically recognize six independent regions on a target sequence for amplification, and the LAMP primers have high specificity. By applying the LAMP primer and the method, the herpes simplex virus I, the herpes simplex virus II, the varicella-zoster virus and the cytomegalovirus can be quickly and accurately detected.
drawings
FIG. 1 is a graph showing LAMP amplification in example 2.
FIG. 2 is a graph of LAMP amplification in example 6.
Detailed Description
the following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
pGEM-T Easy Vector: purchased from promega, product No. a 1360.
example 1 primer design and preparation
A plurality of primers for identifying the herpes simplex virus I, the herpes simplex virus II, the cytomegalovirus and the varicella-zoster virus are obtained by carrying out a great deal of sequence analysis and alignment. And performing a pre-experiment on each primer, and comparing the performances such as sensitivity, specificity and the like to finally obtain four sets of LAMP primer groups for identifying the herpes simplex virus I, the herpes simplex virus II, the varicella-zoster virus and the cytomegalovirus.
The primer group for identifying the herpes simplex virus I type comprises 2 outer primers (F3-1, B3-1), 2 inner primers (FIP-1, BIP-1) and 2 loop primers (LF-1, LB-1), wherein the sequences of the primers are shown as follows (5 '→ 3'):
f3-1 (SEQ ID NO: 1 of the sequence Listing): TGCGGCTCGTGAAGACC, respectively;
b3-1 (SEQ ID NO: 2 of the sequence Listing): TACGGCGACGGTGCGTA, respectively;
FIP-1 (SEQ ID NO: 3 of the sequence listing): TACTTACAGGAGCCCTTGGGACTGGACGGAGATTACACAGCG, respectively;
BIP-1 (sequence 4 of the sequence table): ACCAGCAGGGGGTGACTCTCGGGGATGAAGCGGCT, respectively;
LF-1 (SEQ ID NO: 5 of the sequence Listing): CGGTGCTCCAGGATAAACT, respectively;
LB-1 (SEQ ID NO: 6 of the sequence Listing): TGGACAGCATCGGGGG are provided.
The primer group for identifying the herpes simplex virus II type comprises 2 outer primers (F3-2, B3-2), 2 inner primers (FIP-2, BIP-2) and 2 loop primers (LF-2, LB-2), wherein the sequences of the primers are shown as follows (5 '→ 3'):
f3-2 (SEQ ID NO: 7 of the sequence Listing): TGCCCCATCCGAACGTC, respectively;
b3-2 (SEQ ID NO: 8 of the sequence Listing): CTTCGAGGTGAGGCAGC, respectively;
FIP-2 (SEQ ID NO: 9 of the sequence Listing): CCGCGGTCTCAAAGGCCAGCTTTAGCGCCGTCAGAC, respectively;
BIP-2 (SEQ ID NO: 10 of the sequence Listing): GGCTAGTGAAGATAAACGACAGCGTACTTGCAGGAGGGC, respectively;
LF-2 (SEQ ID NO: 11 of the sequence Listing): AGGAATCCCAGGTTATCCTCTC, respectively;
LB-2 (SEQ ID NO: 12 of the sequence Listing): GACGGAGATCACACAATTTATCTG are provided.
The primer group for identifying varicella-zoster virus comprises 2 outer primers (F3-3, B3-3), 2 inner primers (FIP-3, BIP-3) and 2 loop primers (LF-3, LB-3), wherein the sequences of the primers are shown as follows (5 '→ 3'):
F3-3 (SEQ ID NO: 13 of the sequence Listing): AGGATGAAGACGATGAACC, respectively;
b3-3 (SEQ ID NO: 14 of the sequence Listing): GTTTGGTCTTACGAATCCTC, respectively;
FIP-3 (SEQ ID NO: 15 of the sequence Listing): TTGACGGGGAAGGGGCGTGCGTTTCGGTGGGAAGT, respectively;
BIP-3 (SEQ ID NO: 16 of the sequence listing): CGGATGATTCGGACTCAAATGAGATCGGGACCGCTgATG, respectively;
LF-3 (SEQ ID NO: 17 of the sequence Listing): GACCTGCTGCCTGTAGTTTC, respectively;
LB-3 (SEQ ID NO: 18 of the sequence Listing): ACAAAATATCCAACCGGGATATCGA are provided.
The primer group for identifying the cytomegalovirus comprises 2 outer primers (F3-4, B3-4), 2 inner primers (FIP-4, BIP-4) and 2 loop primers (LF-4, LB-4), wherein the sequences of the primers are shown as follows (5 '→ 3'):
f3-4 (SEQ ID NO: 19 of the sequence Listing): AACAATCCGGCCAGCAC, respectively;
b3-4 (SEQ ID NO: 20 of the sequence Listing): CAGTAACGGCCTACCTG, respectively;
FIP-4 (SEQ ID NO: 21 of the sequence Listing): AGTTGGGCGAGTTAGTCTTGGCTTGCGGGTTCGGTGGTTA, respectively;
BIP-4 (SEQ ID NO: 22 of the sequence Listing): ATAAGCTCAACACTATGGCCGACGAAACAACGCGGGATG, respectively;
LF-4 (SEQ ID NO: 23 of the sequence Listing): GCATACGGGGTACATGTCGA, respectively;
LB-4 (SEQ ID NO: 24 of the sequence Listing): GCTTTACCTGCGGCAACGC are provided.
The primer set for identifying herpes simplex virus type I was designated as primer set I.
The primer set for identifying the herpes simplex virus type II was designated as primer set II.
The primer set for identifying varicella-zoster virus was designated as primer set III.
the primer set for identifying cytomegalovirus was designated as primer set IV.
The primer group I, the primer group II, the primer group III and the primer group IV form a primer combination.
Example 2 detection method establishment
1. Extracting the genome DNA of the virus to be detected or extracting the total DNA of the biological sample to be detected.
2. LAMP amplification was performed using the DNA obtained in step 1 as a template and the primer set I prepared in example 1.
Reaction system of LAMP amplification: 1 μ L10 × ThermoPol Buffer, 1.6 μ L5M betaine, 0.1 μ L50mg/ml BSA, 0.4 μ L100 mM MgSO40.3. mu.L of 20 × Eva Green, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-1, B3-1, FIP-1, BIP-1, LF-1 and LB-1), 2ng of template DNA, using ddH2Make up to 10. mu.L of O. The concentration of each primer in the reaction system is as follows: 0.5. mu. M F3-1, 0.5. mu.MB 3-1, 2. mu.M FIP-1, 2. mu.M BIP-1, 1. mu.M LF-1, 1. mu.M LB-1.
reaction procedure for LAMP amplification: keeping the temperature at 65 ℃ for 50 min. In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
3. LAMP amplification was performed using the DNA obtained in step 1 as a template and the primer set II prepared in example 1.
reaction system of LAMP amplification: 1 μ L10 × ThermoPol Buffer, 1.6 μ L5M betaine, 0.1 μ L50mg/ml BSA, 0.4 μ L100 mM MgSO40.3. mu.L of 20 × Eva Green, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-2, B3-2, FIP-2, BIP-2, LF-2 and LB-2), 2ng of template DNA, using ddH2Make up to 10. mu.L of O. The concentration of each primer in the reaction system is as follows: 0.5. mu. M F3-2, 0.5. mu.MB 3-2, 2. mu.M FIP-2, 2. mu.M BIP-2, 1. mu.M LF-2, 1. mu.M LB-2.
reaction procedure for LAMP amplification: keeping the temperature at 65 ℃ for 50 min. In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
4. LAMP amplification was performed using the DNA obtained in step 1 as a template and the primer set III prepared in example 1.
reaction system of LAMP amplification: 1 μ L10 × ThermoPol Buffer, 1.6 μ L5M betaine, 0.1 μ L50mg/ml BSA, 0.4 μ L100 mM MgSO40.3. mu.L of 20 × Evagreen, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-3, B3-3, FIP-3, BIP-3, LF-3 and LB-3), 2ng of template DNA, using ddH2make up to 10. mu.L of O. The concentration of each primer in the reaction system is as follows: 0.5. mu. M F3-3, 0.5. mu.MB 3-3, 2. mu.M FIP-3, 2. mu.M BIP-3, 1. mu.M LF-3, 1. mu.M LB-3.
reaction procedure for LAMP amplification: keeping the temperature at 65 ℃ for 50 min. In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
5. LAMP amplification was performed using the DNA obtained in step 1 as a template and the primer set IV prepared in example 1.
reaction system of LAMP amplification: 1 μ L10 × ThermoPol Buffer, 1.6 μ L5M betaine, 0.1 μ L50mg/ml BSA, 0.4 μ L100 mM MgSO40.3. mu.L of 20 × EvaGreen, 0.15. mu.L of 100mM dNTPs, 0.4. mu.L of 8U/ml Bst DNA polymerase large fragment, 1. mu.L of primer mix (mixture of F3-4, B3-4, FIP-4, BIP-4, LF-4 and LB-4), 2ng of template DNA, using ddH2Make up to 10. mu.L of O. The concentration of each primer in the reaction system is as follows: 0.5. mu. M F3-4, 0.5. mu.MB 3-4, 2. mu.M FIP-4, 2. mu.M BIP-4, 1. mu.M LF-4, 1. mu.M LB-4.
Reaction procedure for LAMP amplification: keeping the temperature at 65 ℃ for 50 min. In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
if the primer group I is adopted, a positive amplification curve can be obtained, which indicates that the virus to be detected is the herpes simplex virus I; if the primer group II is adopted, a positive amplification curve can be obtained, which indicates that the virus to be detected is the herpes simplex virus II type; if the primer group III is adopted, a positive amplification curve can be obtained, which indicates that the virus to be detected is varicella-zoster virus; if the primer group IV is adopted, a positive amplification curve can be obtained, which indicates that the virus to be detected is cytomegalovirus.
if the primer group I is adopted, a positive amplification curve can be obtained, which indicates that the sample to be detected is infected by the herpes simplex virus I; if the primer group II is adopted, a positive amplification curve can be obtained, which indicates that the sample to be detected is infected by the herpes simplex virus II; if the primer group III is adopted, a positive amplification curve can be obtained, which indicates that the sample to be detected is infected by varicella-zoster virus; if the primer group IV is adopted, a positive amplification curve can be obtained, which indicates that the sample to be detected is infected with cytomegalovirus.
example 3 reference quality particle construction
herpes simplex virus type I reference plasmid: inserting the double-stranded DNA molecule shown in the sequence 25 of the sequence table between ApaI and SacI enzyme cutting sites of a pGEM-T Easy Vector to obtain a reference product plasmid.
Herpes simplex virus type II reference plasmid: inserting the double-stranded DNA molecule shown in the sequence 26 of the sequence table between ApaI and SacI enzyme cutting sites of a pGEM-T Easy Vector to obtain a reference product plasmid.
Varicella-zoster virus reference plasmid: inserting the double-stranded DNA molecule shown in the sequence 27 of the sequence table between ApaI and SacI enzyme cutting sites of a pGEM-T Easy Vector to obtain a reference product plasmid.
cytomegalovirus reference plasmid: inserting the double-stranded DNA molecule shown in the sequence 28 of the sequence table between ApaI and SacI enzyme cutting sites of a pGEM-T Easy Vector to obtain a reference product plasmid.
example 4 specificity
The samples to be tested are respectively as follows: herpes simplex virus I type reference plasmid DNA, herpes simplex virus II type reference plasmid DNA, varicella-zoster virus reference plasmid DNA and cytomegalovirus reference plasmid DNA.
the detection was carried out by the detection method of example 2.
the results are shown in FIG. 1. In fig. 1, the abscissa is the cycle number, and the ordinate is the fluorescence signal intensity, where a is the amplification curve of each sample to be tested when the primer set I is used, B is the amplification curve of each sample to be tested when the primer set II is used, C is the amplification curve of each sample to be tested when the primer set III is used, and D is the amplification curve of each sample to be tested when the primer set IV is used. As can be seen from FIG. 1A, the herpes simplex virus type I reference plasmid DNA gave a positive S-type amplification curve, and the herpes simplex virus type II reference plasmid DNA, the varicella-zoster virus reference plasmid DNA and the cytomegalovirus reference plasmid DNA did not give a negative S-type amplification curve. As can be seen from FIG. 1B, the herpes simplex virus type II reference plasmid DNA gave a positive S-type amplification curve, and the herpes simplex virus type I reference plasmid DNA, the varicella-zoster virus reference plasmid DNA and the cytomegalovirus reference plasmid DNA did not give a negative S-type amplification curve. As can be seen from FIG. 1C, the varicella-zoster virus reference plasmid DNA obtained an S-type amplification curve, the amplification result was positive, and the varicella-zoster virus reference plasmid DNA, the varicella-zoster virus reference plasmid DNA and the cytomegalovirus reference plasmid DNA did not obtain an S-type amplification curve, and the amplification result was negative. The results show that the primer combinations prepared in example 1 all have high specificity.
example 5 sensitivity
The samples to be tested are respectively as follows: herpes simplex virus I type reference plasmid DNA, herpes simplex virus II type reference plasmid DNA, varicella-zoster virus reference plasmid DNA and cytomegalovirus reference plasmid DNA.
1. and (4) diluting each sample to be detected with 10 times of sterile water in a gradient manner to obtain each diluent.
2. each of the dilutions obtained in step 1 was used as a template, and the assay was performed by the assay method of example 2.
due to the different dilution used, the following different reaction systems are formed:
In the reaction system 1, the initial content of the reference plasmid DNA was: 10000 copies;
In the reaction system 2, the initial content of the reference plasmid DNA is: 1000 copies;
In the reaction system 3, the initial content of the reference plasmid DNA is as follows: 500 copies;
In the reaction system 4, the initial content of the reference plasmid DNA is as follows: 100 copies.
Each virus reference plasmid was tested for the 4 lines described above.
The result shows that S-type amplification curves are obtained when the DNA content of herpes simplex virus I-type reference plasmid, herpes simplex virus II-type reference plasmid, varicella-zoster virus reference plasmid and cytomegalovirus reference plasmid is 500-10000 copies, and S-type amplification curves are not obtained when the DNA concentration of herpes simplex virus I-type reference plasmid, herpes simplex virus II-type reference plasmid, varicella-zoster virus reference plasmid and cytomegalovirus reference plasmid is 100 copies. The result shows that the LAMP primer combination for detecting the 4 ophthalmic infectious viruses provided by the invention has higher sensitivity.
Example 6 clinical sample testing
the sample to be tested is: an ocular aspirate sample clinically identified as positive for herpes simplex virus type I, an ocular aspirate sample clinically identified as positive for herpes simplex virus type II, an ocular aspirate sample clinically identified as positive for varicella-zoster virus, an ocular aspirate sample clinically identified as cytomegalovirus, and an ocular aspirate sample of a healthy subject clinically identified as not having herpes simplex virus type I, varicella-zoster virus, or cytomegalovirus.
the detection was carried out by the detection method of example 2.
The results are shown in FIG. 2. In fig. 2, the abscissa is the cycle number, and the ordinate is the fluorescence signal intensity, where a is the amplification curve of each sample to be tested when the primer set I is used, B is the amplification curve of each sample to be tested when the primer set II is used, C is the amplification curve of each sample to be tested when the primer set III is used, and D is the amplification curve of each sample to be tested when the primer set IV is used. As can be seen from FIG. 2A, only the positive sample of herpes simplex virus type I gave an S-type amplification curve (as shown by sequence 25 in the sequence table by sequencing), and none of the positive sample of herpes simplex virus type II, the positive sample of varicella-zoster virus and the positive sample of cytomegalovirus gave an S-type amplification curve, which is consistent with the clinical identification result. As can be seen from FIG. 2B, only the positive sample of herpes simplex virus type II gave an S-type amplification curve (as shown by sequence 26 in the sequence table by sequencing), and none of the positive sample of herpes simplex virus type I, the positive sample of varicella-zoster virus and the positive sample of varicella-zoster virus gave an S-type amplification curve, which is consistent with the clinical identification result. As can be seen from FIG. 2C, only the varicella-zoster virus positive sample obtained an S-type amplification curve (shown by sequence 27 in the sequence table after sequencing), and none of the herpes simplex virus I-type positive sample, the herpes simplex virus II-type positive sample and the cytomegalovirus positive sample obtained an S-type amplification curve, which is consistent with the clinical identification result. As can be seen from FIG. 2D, only the cytomegalovirus positive sample obtained an S-type amplification curve (shown by sequence 28 in the sequence table after sequencing), and none of the herpes simplex virus I-type positive sample, the herpes simplex virus II-type positive sample and the varicella-zoster virus positive sample obtained an S-type amplification curve, which is consistent with the clinical identification result. The results show that the primer combination prepared in the example 1 is used for virus identification, and the results are accurate and reliable. Healthy tissues do not show a positive sigmoid amplification curve, demonstrating that there is no non-specific amplification of the four primer sets for the human normal genome.
Claims (6)
1. the primer combination consists of a primer group I, a primer group II, a primer group III and a primer group IV;
the primer group I consists of a primer F3-1, a primer B3-1, a primer FIP-1, a primer BIP-1, a primer LB-1 and a primer LF-1;
The primer F3-1 is a single-stranded DNA molecule shown in a sequence 1 in a sequence table;
The primer B3-1 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
the primer FIP-1 is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;
the primer BIP-1 is a single-stranded DNA molecule shown in a sequence 4 of a sequence table;
the primer LB-1 is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;
The primer LF-1 is a single-stranded DNA molecule shown in a sequence 6 of a sequence table;
The primer group II consists of a primer F3-2, a primer B3-2, a primer FIP-2, a primer BIP-2, a primer LB-2 and a primer LF-2;
The primer F3-2 is a single-stranded DNA molecule shown in a sequence 7 in a sequence table;
The primer B3-2 is a single-stranded DNA molecule shown in a sequence 8 in a sequence table;
The primer FIP-2 is a single-stranded DNA molecule shown in a sequence 9 of a sequence table;
The primer BIP-2 is a single-stranded DNA molecule shown in a sequence 10 of a sequence table;
the primer LB-2 is a single-stranded DNA molecule shown in a sequence 11 of a sequence table;
the primer LF-3 is a single-stranded DNA molecule shown in a sequence 12 in a sequence table;
The primer group III consists of a primer F3-3, a primer B3-3, a primer FIP-2, a primer BIP-3, a primer LB-3 and a primer LF-3;
the primer F3-3 is a single-stranded DNA molecule shown in a sequence 13 in a sequence table;
the primer B3-3 is a single-stranded DNA molecule shown in a sequence 14 in a sequence table;
The primer FIP-2 is a single-stranded DNA molecule shown in a sequence 15 in a sequence table;
the primer BIP-3 is a single-stranded DNA molecule shown in a sequence 16 in a sequence table;
the primer LB-3 is a single-stranded DNA molecule shown in a sequence 17 of a sequence table;
The primer LF-3 is a single-stranded DNA molecule shown in a sequence 18 in a sequence table;
the primer group IV consists of a primer F3-4, a primer B3-4, a primer FIP-4, a primer BIP-4, a primer LB-4 and a primer LF-4;
the primer F3-4 is a single-stranded DNA molecule shown in a sequence 19 in a sequence table;
The primer B3-4 is a single-stranded DNA molecule shown in a sequence 20 in a sequence table;
the primer FIP-4 is a single-stranded DNA molecule shown in a sequence 21 in a sequence table;
The primer BIP-4 is a single-stranded DNA molecule shown in a sequence 22 of a sequence table;
The primer LB-4 is a single-stranded DNA molecule shown in a sequence 23 of a sequence table;
the primer LF-4 is a single-stranded DNA molecule shown in a sequence 24 of a sequence table.
2. use of the primer combination according to claim 1 for the preparation of a kit for identifying whether a sample to be tested is infected with herpes simplex virus type i, herpes simplex virus type ii, varicella-zoster virus and cytomegalovirus.
3. A kit comprising the primer combination of claim 1; the kit is used for identifying whether a sample to be detected is infected with herpes simplex virus I, herpes simplex virus II, varicella-zoster virus and cytomegalovirus.
4. a method for preparing the kit according to claim 3, comprising the step of packaging each primer individually.
5. a method for identifying whether a virus to be detected is herpes simplex virus I, herpes simplex virus II, varicella-zoster virus or cytomegalovirus comprises the following steps:
(1) extracting the genome DNA of the virus to be detected;
(2) Performing LAMP amplification reaction by using the genomic DNA obtained in the step (1) as a template and respectively using a primer group I, a primer group II, a primer group III and a primer group IV of the primer combination according to claim 1, wherein positive amplification by using the genomic DNA as the template and the virus to be detected as herpes simplex virus I can be realized by using the primer group I, positive amplification by using the genomic DNA as the template and the virus to be detected as herpes simplex virus II can be realized by using the primer group II, positive amplification by using the genomic DNA as the template and the virus to be detected as varicella-zoster virus can be realized by using the primer group III, and positive amplification by using the genomic DNA as the template and the virus to be detected as cytomegalovirus can be realized by using the primer group IV;
the method is a non-disease diagnostic method.
6. a method for assisting in identifying whether a virus to be detected is herpes simplex virus I or herpes simplex virus II or varicella-zoster virus or cytomegalovirus comprises the following steps:
(1) extracting the genome DNA of the virus to be detected;
(2) performing LAMP amplification reaction by using the genomic DNA obtained in the step (1) as a template and respectively using the primer group I, the primer group II, the primer group III and the primer group IV of the primer combination according to claim 1, wherein positive amplification by using the genomic DNA as the template and the virus to be detected as or as a candidate for the herpes simplex virus I type can be realized by using the primer group I, positive amplification by using the genomic DNA as the template and the virus to be detected as or as the candidate for the herpes simplex virus II type can be realized by using the primer group II, positive amplification by using the genomic DNA as the template and the virus to be detected as or as a candidate for the varicella zoster virus can be realized by using the primer group III, positive amplification by using the genomic DNA as the template and the virus to be detected as or as the cytomegalovirus can be realized by using the primer group IV, The primer group III and the primer group IV can not realize positive amplification by taking the genome DNA as a template, and the viruses to be detected are non-herpes simplex virus I type, non-herpes simplex virus II type, non-varicella-zoster virus and non-cytomegalovirus;
The method is a non-disease diagnostic method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610404661.XA CN106048088B (en) | 2016-06-08 | 2016-06-08 | LAMP primer combination for detecting 4 ophthalmic infectious viruses and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610404661.XA CN106048088B (en) | 2016-06-08 | 2016-06-08 | LAMP primer combination for detecting 4 ophthalmic infectious viruses and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106048088A CN106048088A (en) | 2016-10-26 |
| CN106048088B true CN106048088B (en) | 2019-12-13 |
Family
ID=57170623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610404661.XA Active CN106048088B (en) | 2016-06-08 | 2016-06-08 | LAMP primer combination for detecting 4 ophthalmic infectious viruses and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106048088B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754907A (en) * | 2017-01-11 | 2017-05-31 | 博奥生物集团有限公司 | LAMP primer composition and its application for detecting three kinds of bacterial resistance genes of Extended-spectrum β-lactamases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561378A (en) * | 2014-12-24 | 2015-04-29 | 华美生物工程有限公司 | Real-time fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection |
-
2016
- 2016-06-08 CN CN201610404661.XA patent/CN106048088B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561378A (en) * | 2014-12-24 | 2015-04-29 | 华美生物工程有限公司 | Real-time fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection |
Non-Patent Citations (2)
| Title |
|---|
| Rapid Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method;Yoshihiko Enomoto;《JOURNAL OF CLINICAL MICROBIOLOGY》;20050228;第43卷(第2期);951-955 * |
| Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification;Hisatoshi Kaneko;《JOURNAL OF CLINICAL MICROBIOLOGY》;20050731;第43卷(第7期);3290-3296 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106048088A (en) | 2016-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108441580B (en) | Kit for simultaneously detecting HSV-1, HSV-2, VZV herpes viruses by one-step method and detection method | |
| Pfeifer et al. | Cervical trophoblasts for non-invasive single-cell genotyping and prenatal diagnosis | |
| CN109929949B (en) | EBV detection method based on CRISPR-Cas12a and G quadruplex-heme and application thereof | |
| Bispo et al. | Rapid detection and identification of uveitis pathogens by qualitative multiplex real-time PCR | |
| CN112029900A (en) | Rapid nucleic acid detection method and detection system for novel coronavirus | |
| CN106048088B (en) | LAMP primer combination for detecting 4 ophthalmic infectious viruses and application | |
| WO2018151339A1 (en) | Method for detecting target gene, using dcas9/grna complex and fluorescence marker | |
| CN106987657B (en) | Primer combination for identifying bovine virus diarrhea virus and bovine rotavirus and application thereof | |
| CN105861727A (en) | LAMP primer combination for detecting three ophthalmic infection spirochetes and application | |
| Chisanga et al. | Evidence for placental HPV infection in both HIV positive and negative women | |
| US20150376725A1 (en) | HPV Detection in Urine | |
| CN114085929B (en) | Kit for detecting African swine fever virus wild strain and vaccine strain | |
| CN113234866B (en) | Detection kit for synchronously detecting pathogens of multiple blood circulation systems and detection method thereof | |
| CN115976029A (en) | Aptamer specifically binding to GDF15 and application thereof | |
| CN111500768B (en) | Primer probe for identifying novel coronavirus and application of primer probe in dual-digital PCR | |
| CN116240312A (en) | Novel composition for combined detection of coronavirus, HIV and monkey pox virus | |
| JP3494509B2 (en) | Nucleic acid synthesis method | |
| US6268127B1 (en) | Method for preparing DNA from serum and plasma | |
| CN108950064B (en) | Kit and method for detecting EBV infection in ocular trace biological sample | |
| WO2019075233A1 (en) | Methods for the rapid detection and identification of uveitis pathogens | |
| CN107385116B (en) | Method for detecting type 1 human immunodeficiency virus and special reagent set thereof | |
| CN111363741B (en) | Primer group for detecting human ocular adenovirus and application thereof | |
| CN112322782B (en) | Primer group, kit and method for quickly detecting KSHV through polymerase helix reaction | |
| Al-Salami et al. | Molecular and Immunological Study for Detection of BK Human Polyoma Virus in Patient with Hemorrhagic Cystitis in some Iraq People | |
| CN108220476B (en) | Kit for detecting varicella-zoster virus and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210517 Address after: 101300 second floor, building 37, lanxiyuan District 3, Renhe Town, Shunyi District, Beijing Patentee after: BEIJING ZHIDE MEDICAL LABORATORY Co.,Ltd. Address before: 8 worker's Stadium South Road, Chaoyang District, Beijing 100020 Patentee before: BEIJING CHAO-YANG HOSPITAL, CAPITAL MEDICAL University |
|
| TR01 | Transfer of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Lamp primer combination for detecting four ophthalmic infectious viruses and its application Effective date of registration: 20220114 Granted publication date: 20191213 Pledgee: Zhongguancun Beijing technology financing Company limited by guarantee Pledgor: BEIJING ZHIDE MEDICAL LABORATORY CO.,LTD. Registration number: Y2022990000023 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220610 Granted publication date: 20191213 Pledgee: Zhongguancun Beijing technology financing Company limited by guarantee Pledgor: BEIJING ZHIDE MEDICAL LABORATORY CO.,LTD. Registration number: Y2022990000023 |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221027 Address after: 214028 A601-3, 530 Building, No. 18 Qingyuan Road, Xinwu District, Wuxi, Jiangsu Province Patentee after: Zhide Technology (Wuxi) Co.,Ltd. Address before: 101300 second floor, building 37, lanxiyuan District 3, Renhe Town, Shunyi District, Beijing Patentee before: BEIJING ZHIDE MEDICAL LABORATORY CO.,LTD. |