CN109991413A - 一种猫杯状病毒荧光定量检测卡及检测方法 - Google Patents
一种猫杯状病毒荧光定量检测卡及检测方法 Download PDFInfo
- Publication number
- CN109991413A CN109991413A CN201711495378.3A CN201711495378A CN109991413A CN 109991413 A CN109991413 A CN 109991413A CN 201711495378 A CN201711495378 A CN 201711495378A CN 109991413 A CN109991413 A CN 109991413A
- Authority
- CN
- China
- Prior art keywords
- feline calicivirus
- quantitative detection
- reaction film
- detection
- bonding pad
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 64
- 241000714201 Feline calicivirus Species 0.000 title claims abstract description 46
- 238000006243 chemical reaction Methods 0.000 claims abstract description 32
- 239000004816 latex Substances 0.000 claims abstract description 23
- 229920000126 latex Polymers 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 10
- 241001494479 Pecora Species 0.000 claims abstract description 8
- 230000008878 coupling Effects 0.000 claims abstract description 7
- 238000010168 coupling process Methods 0.000 claims abstract description 7
- 238000005859 coupling reaction Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 6
- 210000000214 mouth Anatomy 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 210000003928 nasal cavity Anatomy 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 2
- 238000004393 prognosis Methods 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 8
- 239000006185 dispersion Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 6
- 241000700605 Viruses Species 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 208000003322 Coinfection Diseases 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 101000768957 Acholeplasma phage L2 Uncharacterized 37.2 kDa protein Proteins 0.000 description 1
- 101000823746 Acidianus ambivalens Uncharacterized 17.7 kDa protein in bps2 3'region Proteins 0.000 description 1
- 101000916369 Acidianus ambivalens Uncharacterized protein in sor 5'region Proteins 0.000 description 1
- 101000769342 Acinetobacter guillouiae Uncharacterized protein in rpoN-murA intergenic region Proteins 0.000 description 1
- 101000823696 Actinobacillus pleuropneumoniae Uncharacterized glycosyltransferase in aroQ 3'region Proteins 0.000 description 1
- 101000786513 Agrobacterium tumefaciens (strain 15955) Uncharacterized protein outside the virF region Proteins 0.000 description 1
- 101000618005 Alkalihalobacillus pseudofirmus (strain ATCC BAA-2126 / JCM 17055 / OF4) Uncharacterized protein BpOF4_00885 Proteins 0.000 description 1
- 102100020724 Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Human genes 0.000 description 1
- 101000967489 Azorhizobium caulinodans (strain ATCC 43989 / DSM 5975 / JCM 20966 / LMG 6465 / NBRC 14845 / NCIMB 13405 / ORS 571) Uncharacterized protein AZC_3924 Proteins 0.000 description 1
- 101000823761 Bacillus licheniformis Uncharacterized 9.4 kDa protein in flaL 3'region Proteins 0.000 description 1
- 101000819719 Bacillus methanolicus Uncharacterized N-acetyltransferase in lysA 3'region Proteins 0.000 description 1
- 101000789586 Bacillus subtilis (strain 168) UPF0702 transmembrane protein YkjA Proteins 0.000 description 1
- 101000792624 Bacillus subtilis (strain 168) Uncharacterized protein YbxH Proteins 0.000 description 1
- 101000790792 Bacillus subtilis (strain 168) Uncharacterized protein YckC Proteins 0.000 description 1
- 101000819705 Bacillus subtilis (strain 168) Uncharacterized protein YlxR Proteins 0.000 description 1
- 101000948218 Bacillus subtilis (strain 168) Uncharacterized protein YtxJ Proteins 0.000 description 1
- 101000718627 Bacillus thuringiensis subsp. kurstaki Putative RNA polymerase sigma-G factor Proteins 0.000 description 1
- 101000641200 Bombyx mori densovirus Putative non-structural protein Proteins 0.000 description 1
- 241000714198 Caliciviridae Species 0.000 description 1
- 208000006339 Caliciviridae Infections Diseases 0.000 description 1
- 101710193695 Capsid protein precursor Proteins 0.000 description 1
- 101000947633 Claviceps purpurea Uncharacterized 13.8 kDa protein Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101000948901 Enterobacteria phage T4 Uncharacterized 16.0 kDa protein in segB-ipI intergenic region Proteins 0.000 description 1
- 101000805958 Equine herpesvirus 4 (strain 1942) Virion protein US10 homolog Proteins 0.000 description 1
- 101000790442 Escherichia coli Insertion element IS2 uncharacterized 11.1 kDa protein Proteins 0.000 description 1
- 101000788354 Escherichia phage P2 Uncharacterized 8.2 kDa protein in gpA 5'region Proteins 0.000 description 1
- 101000770304 Frankia alni UPF0460 protein in nifX-nifW intergenic region Proteins 0.000 description 1
- 101000797344 Geobacillus stearothermophilus Putative tRNA (cytidine(34)-2'-O)-methyltransferase Proteins 0.000 description 1
- 101000748410 Geobacillus stearothermophilus Uncharacterized protein in fumA 3'region Proteins 0.000 description 1
- 101000772675 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) UPF0438 protein HI_0847 Proteins 0.000 description 1
- 101000631019 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Uncharacterized protein HI_0350 Proteins 0.000 description 1
- 101000768938 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 8.9 kDa protein in int-C1 intergenic region Proteins 0.000 description 1
- 101000785414 Homo sapiens Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Proteins 0.000 description 1
- 101000833492 Homo sapiens Jouberin Proteins 0.000 description 1
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 description 1
- 102100024407 Jouberin Human genes 0.000 description 1
- 101000782488 Junonia coenia densovirus (isolate pBRJ/1990) Putative non-structural protein NS2 Proteins 0.000 description 1
- 101000811523 Klebsiella pneumoniae Uncharacterized 55.8 kDa protein in cps region Proteins 0.000 description 1
- 101000818409 Lactococcus lactis subsp. lactis Uncharacterized HTH-type transcriptional regulator in lacX 3'region Proteins 0.000 description 1
- 101000878851 Leptolyngbya boryana Putative Fe(2+) transport protein A Proteins 0.000 description 1
- 101000758828 Methanosarcina barkeri (strain Fusaro / DSM 804) Uncharacterized protein Mbar_A1602 Proteins 0.000 description 1
- 101001122401 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF3 Proteins 0.000 description 1
- 101001055788 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) Pentapeptide repeat protein MfpA Proteins 0.000 description 1
- 101000740670 Orgyia pseudotsugata multicapsid polyhedrosis virus Protein C42 Proteins 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- 241000282373 Panthera pardus Species 0.000 description 1
- 241000282376 Panthera tigris Species 0.000 description 1
- 206010034960 Photophobia Diseases 0.000 description 1
- 101000769182 Photorhabdus luminescens Uncharacterized protein in pnp 3'region Proteins 0.000 description 1
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 1
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 1
- 101000961392 Pseudescherichia vulneris Uncharacterized 29.9 kDa protein in crtE 3'region Proteins 0.000 description 1
- 101000731030 Pseudomonas oleovorans Poly(3-hydroxyalkanoate) polymerase 2 Proteins 0.000 description 1
- 101001065485 Pseudomonas putida Probable fatty acid methyltransferase Proteins 0.000 description 1
- 101000711023 Rhizobium leguminosarum bv. trifolii Uncharacterized protein in tfuA 3'region Proteins 0.000 description 1
- 101000948156 Rhodococcus erythropolis Uncharacterized 47.3 kDa protein in thcA 5'region Proteins 0.000 description 1
- 101000917565 Rhodococcus fascians Uncharacterized 33.6 kDa protein in fasciation locus Proteins 0.000 description 1
- 101000790284 Saimiriine herpesvirus 2 (strain 488) Uncharacterized 9.5 kDa protein in DHFR 3'region Proteins 0.000 description 1
- 101000936719 Streptococcus gordonii Accessory Sec system protein Asp3 Proteins 0.000 description 1
- 101000788499 Streptomyces coelicolor Uncharacterized oxidoreductase in mprA 5'region Proteins 0.000 description 1
- 101001102841 Streptomyces griseus Purine nucleoside phosphorylase ORF3 Proteins 0.000 description 1
- 101000708557 Streptomyces lincolnensis Uncharacterized 17.2 kDa protein in melC2-rnhH intergenic region Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 101000649826 Thermotoga neapolitana Putative anti-sigma factor antagonist TM1081 homolog Proteins 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 101000827562 Vibrio alginolyticus Uncharacterized protein in proC 3'region Proteins 0.000 description 1
- 101000778915 Vibrio parahaemolyticus serotype O3:K6 (strain RIMD 2210633) Uncharacterized membrane protein VP2115 Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000002788 crimping Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 239000007970 homogeneous dispersion Substances 0.000 description 1
- 238000007373 indentation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Virology (AREA)
- Urology & Nephrology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种猫杯状病毒荧光定量检测卡及检测方法,所述荧光定量检测卡由加样槽、扫描窗及手持装置组成,所述加样槽覆盖在样品垫、结合垫及部分反应膜上方,所述扫描窗覆盖在反应膜上方,所述结合垫包被有荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体,所述反应膜含有检测线及质控线,所述检测线包被有抗猫杯状病毒单克隆抗体,所述质控线包被有羊抗鼠多克隆抗体。本发明可提供定量检测结果,此外,本发明公开了一种基于定量检测结果的猫杯状病毒病情及预后判断方法。本发明可定量检测猫杯状病毒,具有灵敏度高、易操作、稳定性好等优点,可实现对临床样本的跟踪监测。
Description
技术领域
本发明涉及一种猫杯状病毒荧光定量检测卡及检测方法,属于生物检测领域。
背景技术
猫杯状病毒(Feline calicivirus,FCV)属于杯状病毒科,疱疹病毒属,是一种单链RNA 病毒,呈正二十面体对称。可引起猫及猫科动物的口腔及呼吸道传染病,也可引起结膜炎,猫杯状病毒也被命名为猫传染性鼻-结膜炎病毒。感染该病毒后,患病动物可出现口腔溃疡、眼鼻分泌物增多、羞明、气管炎和支气管炎等症状。1957年Fastier首次分离出该病毒,此后国内科学家也从病猫中分离出该病毒。FCV基因组全长7690bp,编码3个开放阅读框(ORF), ORF1编码非结构蛋白,ORF2编码衣壳蛋白前体,剩余的ORF3编码一种功能未知的蛋白。猫杯状病毒不仅感染猫,也可感染豹子、东北虎及非洲狮等猫科动物。猫杯状病毒单独感染一般不会致命,但如果和其他病原混合感染将会引起致死性疾病。
FCV常和其他病毒和寄生虫等混合感染,且病毒自身较容易变异,导致FCV检测较为困难。目前FCV的检测方法包括血清中和实验、琼脂扩散实验、荧光定量PCR、ELISA及胶体金等。这些检测方法需要专业的知识及专业的仪器设备,且对检测人员要求较高,得到的结果需要进行复杂的整理,耗时耗力。
发明内容
本发明的目的在于提供一种操作简单、结果易呈现且不需要专业仪器设备和操作人员的猫杯状病毒荧光定量检测卡,实现对猫杯状病毒及时、快速及高灵敏度地检测。
本发明所提供的一种猫杯状病毒荧光定量检测卡,其特征在于,包括加样槽、扫描窗及手持装置,所述加样槽覆盖在样品垫、结合垫及部分反应膜上方,所述扫描窗覆盖在反应膜上方,所述结合垫包被有荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体,所述反应膜含有检测线及质控线,所述检测线包被有抗猫杯状病毒单克隆抗体,所述质控线包被有羊抗鼠多克隆抗体。
所述检测卡可对样本中猫杯状病毒的含量进行定量检测,赋予其特定的AU值。
所述检测卡的待检测物为用拭子沾取的猫科动物的粪便及鼻腔、口腔、眼分泌物或血清。
所述加样槽下方样品垫、结合垫及反应膜的组装顺序为:结合垫上方2~3mm压接在反应膜下端,样品垫压接在剩余结合垫下端。
所述加样槽的形状为椭圆形,可容纳150μL液体。
所述检测线包被的抗猫杯状病毒单克隆抗体不同于结合垫上包被的荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体,且检测线包被的抗猫杯状病毒单克隆抗体需要由溶解有0.5%蔗糖的硼酸缓冲溶液稀释。
所述质控线包被的羊抗鼠多克隆抗体需要由硼酸缓冲溶液稀释。
一种猫杯状病毒荧光定量检测卡的制备方法,其特征在于,由以下步骤组成:
(1)将荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体均匀包被在结合垫上,于37℃烘箱中烘干备用;
(2)将稀释的抗猫杯状病毒单克隆抗体均匀包被在反应膜的检测线上,于37℃烘箱中烘干备用;
(3)将稀释的羊抗鼠多克隆抗体均匀包被在反应膜的检测线上,于37℃烘箱中烘干备用;
(4)将制备好的结合垫、样品垫和反应膜组装成包含加样槽、扫描窗和手持装置的检测卡。
本发明提供了一种猫杯状病毒的定量及赋值方法,其特征在于,该方法包括以下步骤:
(1)制备不同含量的猫杯状病毒抗原校准品(0-500AU);
(2)将步骤(1)中稀释好的疫苗加至加样槽中,10min后检测其扫描窗中检测线的荧光值;
(3)将校准品及对应荧光值做线性拟合;
(4)临床样本测试时将测得的检测线荧光值带入拟合函数,计算所对应的AU值。
本发明依据检验结果,提供了一种猫杯状病毒病情及预后的判断方法,其特征在于:
0-20AU:阴性
20-50AU:疑似携带
>50AU:阳性
与现有技术相比,本发明具有如下有益效果:
(1)本发明基于荧光定量侧向层析技术,一步加样后即可得到定量数值,操作简便。
(2)本发明的检测样本来源于患病动物的粪便、分泌物及血清,无需特殊处理,样本来源广,操作简单。
(3)本发明可对样本中的猫杯状病毒含量进行定量测定,结果较为直观。
(4)本发明提供了一种基于定量数值的猫杯状病毒感染病情及预后判断方法。
附图说明
图1显示了本发明的猫杯状病毒荧光定量检测卡的结构示意图;其中,1指示加样槽,其覆盖在样品垫、结合垫及部分反应膜上方;2指示扫描窗,其覆盖在反应膜上方;3指示手持区;4指示质控线;5指示检测线。
具体实施方式
下面结合具体实施例对本发明做进一步说明,但这些实施例仅是范例性的,并不限制本发明的范围。
实施例1.荧光乳胶微球的制备方法,包括以下步骤:
1.荧光乳胶微球的活化
(A)将定量的荧光乳胶微球溶液加到磷酸盐缓冲溶液中,混合均匀后在16000rpm下,离心20min后移去上清液,加入磷酸盐缓冲溶液,超声60s,得到荧光乳胶微球分散液。
(B)向步骤(A)中的荧光乳胶微球分散液中迅速加入EDC&NHS活化剂,于涡旋振荡器上混合均匀后,室温下震荡活化30min。
2.荧光乳胶微球的偶联
(C)将步骤(B)中活化好的荧光乳胶微球分散液在16000rpm下,离心20min后移去上清液,用步骤(A)中的缓冲溶液清洗,再次以相同条件离心后弃去上清液,用步骤(D) 中的缓冲溶液复溶重悬。
(D)将猫杯状病毒抗原加入缓冲溶液中,使其浓度稀释至0.34mg/mL
(E)将步骤(C)中复溶的荧光乳胶微球分散液加入到步骤(D)中的抗体稀释液中,在涡旋震荡器上混合均匀,超声60s,然后在室温下震荡反应3h。
3.荧光乳胶微球的封闭
(F)反应结束后,将步骤(E)中偶联好的荧光乳胶微球分散液超声60s,在16000rpm下,离心20min后移去上清液,加入封闭液,然后固定在涡旋振荡器上在室温下震荡反应12h。
4.荧光乳胶微球的重悬
(G)将步骤(F)中封闭好的荧光乳胶微球分散液,在16000rpm下,离心20min后移去上清液,再次加入等量的封闭液,混合均匀后再次在相同条件下离心。
(H)离心后,弃去步骤(G)中溶液的上清液,向其中加入微球重悬液,吹打均匀,得到制备好的荧光乳胶微球液分散液。
实施例2.一种猫杯状病毒荧光定量检测卡的制作方法,包括以下步骤:
(1)将结合垫浸泡于溶有BSA的磷酸盐处理液中,干燥后将制备好的荧光乳胶微球分散液均匀喷涂于结合垫上,于烘箱中干燥。
(2)将稀释至2mg/mL的抗猫杯状病毒单克隆抗体以1.6μL/cm的速度喷划于反应膜的检测线上,干燥。
(3)将稀释至2.6mg/mL的羊抗鼠多克隆抗体以1.2μL/cm的速度喷划于反应膜的质控线上,干燥。
(4)将喷涂有荧光乳胶微球的结合垫裁切下来,将前端的2~3mm粘贴覆盖在反应膜上。
(5)将样品垫浸泡于磷酸盐处理液中,干燥,将裁切成宽度为22.5mm的样品垫覆盖在反应膜的后端,且与反应膜下缘平行。
(6)将粘贴好结合垫、样品垫及反应膜的底板切成宽度为3.95mm的试纸条,将试纸条压入卡壳的下盖中,盖上上盖,组装成包含加样槽、扫描窗和手持装置的猫杯状病毒荧光定量检测卡。
步骤(6)中的底板为PVC底板。
步骤(6)中的卡壳为塑料卡壳。
实施例3.一种检测猫杯状病毒的方法,包括以下操作步骤:
(1)用拭子沾取待检动物的粪便及鼻腔、口腔、眼分泌物或血清,将沾取物溶解于稀释液中;
(2)将步骤(1)中的稀释液加至加样槽中,使其逐渐渗入样品垫和结合垫,最终将偶联有抗猫杯状病毒单克隆抗体的荧光乳胶微球渗透至反应膜,使得反应膜的检测线和质控线可以捕获荧光微球;
(3)将检测卡插入配套定量检测仪器中,仪器对扫描窗下的反应膜自动扫描,最终获得稀释液中猫杯状病毒的具体含量;
(4)分析检测结果,生成检测报告,判断患病动物的带毒情况并做进一步的诊断处理。
实施例4.检测卡性能评估
1.稳定性测定
将组装好的检测卡分别放置在4℃及50℃环境下做加速变性实验,每隔7d、14d及21d 用试纸条对高、中、低三个浓度水平的样本进行检测,结果显示,三种浓度水平样本的检测CV值分别为4%、6%、6.5%,符合美国临床化学会对临床样本检测稳定性的要求。
2.准确性测定
将猫杯状病毒荧光定量检测卡成品在宠物医院临床检测,检测对象分为健康动物和患病动物,结果显示,95%的健康动物的检测值小于20AU,处于正常值的设定范围,92%的患病动物的检测值大于50AU,超过正常值的设定范围,说明本检测卡准确性较好,能应用于临床中对猫杯状病毒的即时检测。
Claims (10)
1.一种猫杯状病毒荧光定量检测卡,其特征在于,包括加样槽、扫描窗及手持装置,所述加样槽覆盖在样品垫、结合垫及部分反应膜上方,所述扫描窗覆盖在反应膜上方,所述结合垫包被有荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体,所述反应膜含有检测线及质控线,所述检测线包被有抗猫杯状病毒单克隆抗体,所述质控线包被有羊抗鼠多克隆抗体。
2.根据权利要求1所述的一种猫杯状病毒荧光定量检测卡,其特征在于,所述检测卡可对样本中猫杯状病毒的含量进行定量检测,赋予其特定的AU值。
3.根据权利要求1所述的一种猫杯状病毒荧光定量检测卡,其特征在于,所述检测卡的待检测物为用拭子沾取的猫科动物的粪便及鼻腔、口腔、眼分泌物或血清。
4.根据权利要求1所述的一种猫杯状病毒荧光定量检测卡,其特征在于,所述加样槽下方样品垫、结合垫及反应膜的组装顺序为:结合垫上方2~3mm压接在反应膜下端,样品垫压接在剩余结合垫下端。
5.根据权利要求1所述的一种猫杯状病毒荧光定量检测卡,其特征在于所述加样槽的形状为椭圆形,可容纳150μL液体。
6.根据权利要求1所述的一种猫杯状病毒荧光定量检测卡,其特征在于,所述检测线包被的抗猫杯状病毒单克隆抗体不同于结合垫上包被的荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体。
7.根据权利要求1所述的一种猫杯状病毒荧光定量检测卡,其特征在于,所述质控线包被的羊抗鼠多克隆抗体需要由硼酸缓冲溶液稀释。
8.一种猫杯状病毒荧光定量检测卡的制备方法,其特征在于,由以下步骤组成:
(1)将荧光乳胶微球偶联的抗猫杯状病毒单克隆抗体均匀包被在结合垫上,于37℃烘箱中烘干备用;
(2)将稀释的抗猫杯状病毒单克隆抗体均匀包被在反应膜的检测线上,于37℃烘箱中烘干备用;
(3)将稀释的羊抗鼠多克隆抗体均匀包被在反应膜的质控线上,于37℃烘箱中烘干备用;
(4)将制备好的结合垫、样品垫和反应膜组装成包含加样槽、扫描窗和手持装置的检测卡。
9.一种猫杯状病毒的定量及赋值方法,其特征在于,该方法包括以下步骤:
(1)制备不同含量的猫杯状病毒抗原校准品(0-500AU);
(2)将步骤(1)中稀释好的疫苗加至加样槽中,10min后检测其扫描窗中检测线的荧光值;
(3)将校准品及对应荧光值做线性拟合;
(4)临床样本测试时将测得的检测线荧光值带入拟合函数,计算所对应的AU值。
10.本发明依据检验结果,提供了一种猫杯状病毒病情及预后的判断方法,其特征在于:
0-20AU:阴性
20-50AU:疑似携带
>50AU:阳性。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711495378.3A CN109991413A (zh) | 2017-12-31 | 2017-12-31 | 一种猫杯状病毒荧光定量检测卡及检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711495378.3A CN109991413A (zh) | 2017-12-31 | 2017-12-31 | 一种猫杯状病毒荧光定量检测卡及检测方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN109991413A true CN109991413A (zh) | 2019-07-09 |
Family
ID=67111727
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201711495378.3A Pending CN109991413A (zh) | 2017-12-31 | 2017-12-31 | 一种猫杯状病毒荧光定量检测卡及检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109991413A (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112321722A (zh) * | 2020-11-13 | 2021-02-05 | 杭州亿米诺生物科技有限公司 | 一种猫杯状病毒vp1-vp2重组蛋白及其制备方法和应用 |
CN113817726A (zh) * | 2021-11-22 | 2021-12-21 | 苏州蝌蚪生物技术有限公司 | 用于检测猫杯状病毒的扩增引物组合物及试剂盒 |
CN114839378A (zh) * | 2022-07-04 | 2022-08-02 | 山东康华生物医疗科技股份有限公司 | 一种猫疱疹和猫杯状病毒联合检测试剂盒 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2184567C1 (ru) * | 2001-05-16 | 2002-07-10 | Федеральное государственное учреждение Всероссийский государственный научно-исследовательский институт контроля, стандартизации и сертификации ветеринарных препаратов - центр качества ветеринарных препаратов и кормов | Штамм feline calicivirus, используемый для контроля иммуногенной активности вакцин, изготовления специфических лечебно-профилактических и диагностических биопрепаратов |
CN106053801A (zh) * | 2016-06-01 | 2016-10-26 | 中国农业科学院哈尔滨兽医研究所 | 用于检测猫杯状病毒IgG抗体的ELISA试剂盒及其检测方法 |
CN106841608A (zh) * | 2016-12-20 | 2017-06-13 | 江苏雷森生物科技有限公司 | 一种快速确诊猫白血病的检测方法及其试纸条的制备 |
CN107132348A (zh) * | 2017-02-22 | 2017-09-05 | 江苏雷森生物科技有限公司 | 一种彩色荧光颗粒标记抗体的方法及使用其制备的试纸条 |
-
2017
- 2017-12-31 CN CN201711495378.3A patent/CN109991413A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2184567C1 (ru) * | 2001-05-16 | 2002-07-10 | Федеральное государственное учреждение Всероссийский государственный научно-исследовательский институт контроля, стандартизации и сертификации ветеринарных препаратов - центр качества ветеринарных препаратов и кормов | Штамм feline calicivirus, используемый для контроля иммуногенной активности вакцин, изготовления специфических лечебно-профилактических и диагностических биопрепаратов |
CN106053801A (zh) * | 2016-06-01 | 2016-10-26 | 中国农业科学院哈尔滨兽医研究所 | 用于检测猫杯状病毒IgG抗体的ELISA试剂盒及其检测方法 |
CN106841608A (zh) * | 2016-12-20 | 2017-06-13 | 江苏雷森生物科技有限公司 | 一种快速确诊猫白血病的检测方法及其试纸条的制备 |
CN107132348A (zh) * | 2017-02-22 | 2017-09-05 | 江苏雷森生物科技有限公司 | 一种彩色荧光颗粒标记抗体的方法及使用其制备的试纸条 |
Non-Patent Citations (1)
Title |
---|
王一鸣等: "猫传染性鼻-结膜炎实验室诊断方法研究进展", 《中国兽医杂志》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112321722A (zh) * | 2020-11-13 | 2021-02-05 | 杭州亿米诺生物科技有限公司 | 一种猫杯状病毒vp1-vp2重组蛋白及其制备方法和应用 |
CN113817726A (zh) * | 2021-11-22 | 2021-12-21 | 苏州蝌蚪生物技术有限公司 | 用于检测猫杯状病毒的扩增引物组合物及试剂盒 |
CN114839378A (zh) * | 2022-07-04 | 2022-08-02 | 山东康华生物医疗科技股份有限公司 | 一种猫疱疹和猫杯状病毒联合检测试剂盒 |
CN114839378B (zh) * | 2022-07-04 | 2022-09-06 | 山东康华生物医疗科技股份有限公司 | 一种猫疱疹和猫杯状病毒联合检测试剂盒 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Flynn et al. | Influenza D virus in cattle, Ireland | |
CN106290867B (zh) | 一种同时检测五种禽垂直传播疾病抗体的液相芯片及方法 | |
CN109991413A (zh) | 一种猫杯状病毒荧光定量检测卡及检测方法 | |
JP2019148610A (ja) | 凝集の検出および測定のための方法 | |
JP2020128999A5 (zh) | ||
CN111233985A (zh) | 一种新型冠状病毒IgA抗体快速检测试纸条的制备方法 | |
CN105527430A (zh) | 一种直接检测肠道病毒71型和柯萨奇病毒a16型的试剂盒及其应用 | |
CN111474346A (zh) | 猪流行性腹泻病毒IgA和IgG抗体检测试剂盒及其制备方法和应用 | |
US7067258B2 (en) | Human papillomavirus multiplexed assay | |
CN103308688A (zh) | 猪圆环病毒2型IgM抗体ELISA检测试剂盒及制备方法 | |
CN203838160U (zh) | 猪伪狂犬病毒gB和gE抗体的ELISA检测试剂盒 | |
CN112067803A (zh) | 磁性纳米颗粒标记的免疫层析法制备的新型冠状病毒检测试剂盒 | |
CN108918866A (zh) | 一种炎症标志物poct联合检测试剂盒套装 | |
CN109374887A (zh) | 牛病毒性腹泻病毒抗原胶体金检测试剂盒及其应用 | |
CN203759021U (zh) | 诺如病毒检测试剂盒 | |
CN112379089A (zh) | 一种基于量子点微球免疫层析试纸条的新冠病毒检测方法 | |
Moennig et al. | Classical swine fever | |
CN111007251A (zh) | 纳米酶试纸条检测诺如病毒的方法 | |
CN104360055A (zh) | 测定水痘-带状疱疹病毒中和抗体的试剂盒及生产方法 | |
CN104531895B (zh) | 一种测定病毒滴度的方法 | |
Sarkar et al. | Development of a cell-elisa using anti-nucleocapsid protein monoclonal antibody for the titration of peste des petits ruminants (PPR) vaccine virus | |
Saushkin et al. | A new sampling format for the diagnostics of bovine infectious diseases in dried blood spots by ELISA | |
Pan et al. | A poly (dimethylsiloxane)-based solid-phase microchip platform for dual detection of Pseudorabies virus gD and gE antibodies | |
Kimura et al. | A highly quantitative detection system for cell entry of human norovirus-like particles based on the complementation of NanoLuc luciferase | |
CN105759029A (zh) | 麻疹病毒检测试剂盒及使用方法 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190709 |
|
WD01 | Invention patent application deemed withdrawn after publication |