CN109142726A - A kind of gold label test strip and its preparation and application for campylobacter jejuni detection - Google Patents
A kind of gold label test strip and its preparation and application for campylobacter jejuni detection Download PDFInfo
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- CN109142726A CN109142726A CN201811008658.1A CN201811008658A CN109142726A CN 109142726 A CN109142726 A CN 109142726A CN 201811008658 A CN201811008658 A CN 201811008658A CN 109142726 A CN109142726 A CN 109142726A
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Abstract
The invention belongs to technical field of immunoassay, and in particular to a kind of gold label test strip and its preparation and application for campylobacter jejuni detection.Test strips of the invention are detection target proteins with the CjaA albumen of campylobacter jejuni, the assembling that test strips are carried out using the monoclonal antibody of two kinds of identifying purpose albumen different epitopes as gold conjugated antibody and detection antibody, for carrying out on-site test to poultry surface wipes sample.The test strips are portable quickly, and not by the interference of environmental condition, detecting duration is only needed 10 minutes；With non-campylobacter jejuni no cross reaction；High sensitivity, the concentrations of minimum campylobacter jejuni are 100 CFU/ μ L.The field quick detection of poultry sample jejuni can be achieved in the present invention, is suitable for the units such as production of poultry meat processing enterprise, inspection and quarantine.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of gold label test strip for campylobacter jejuni detection
And its preparation and application.
Background technique
Campylobacter jejuni (Campylobacter jejuni) is an important people beast after detection of Salmonella, shigella dysenteriae
Illness opportunistic pathogen altogether, can lead to enterogastritis and food poisoning, is forerunner's factor of mankind's actue infectious polyradiculoneuritis, the mankind are mainly led to
It crosses and eats by the food of its pollution and illness.Campylobacter jejuni is usually colonized in the enteron aisle of poultry, and is discharged with excrement, in fowl
Meat the links such as to butcher, process, transporting, selling popular on a large scale, it is seriously polluted and inevitable.It is quickly detected from fowl in time
The campylobacter jejuni on meat surface examines inspection for the harm prevention and control of foodborne bacterial pathogens, public health, animal and veterinary and entry and exit
Epidemic disease is significant.The condition of culture of campylobacter jejuni is harsh, and conventional bacteria cultivation is the national standard method for detecting campylobacter jejuni,
But there are cumbersome, the specific drawbacks such as not strong, time-consuming and laborious；Though and using PCR as the molecular biology for detection of representative
Right high sensitivity, high specificity, but need to be to rely on laboratory, testing result is easy to appear false positive, and being not suitable for scene makes
With.Quick, sensitive, special, the accurate campylobacter jejuni visualization real-time detection method of one kind is researched and developed to poultry and birds meat products
Security monitoring and the epidemiological survey of foodborne bacterial pathogens are significant.
Summary of the invention
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide one kind to be used for campylobacter jejuni
The gold label test strip of detection.The test strips can efficiently detect in poultry surface wipes sample in production of poultry meat processing site
Campylobacter jejuni.Test strips of the invention are detection target proteins with the CjaA albumen of campylobacter jejuni, by two kinds of identification mesh
Albumen different epitopes monoclonal antibody respectively as gold conjugated antibody and detection antibody carry out test strips assembling, use
In to poultry surface wipes sample progress on-site test.The test strips are portable quickly, not by the interference of environmental condition, detect duration
Only need 10 minutes；With non-campylobacter jejuni no cross reaction；The concentrations of high sensitivity, minimum campylobacter jejuni are
100CFU/μL.The field quick detection of poultry sample jejuni can be achieved in the present invention, is suitable for production of poultry meat processing enterprise
The units such as industry, inspection and quarantine.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
The first aspect of the present invention, provide it is a kind of for campylobacter jejuni detection gold label test strip, including support plate, with
And sample pad, gold-labelled pad, nitrocellulose filter detection layers and the water absorption pad being arranged successively from sample-adding end positioned at support plate surface,
Include the campylobacter jejuni monoclonal antibody I of colloid gold label in the gold-labelled pad, is wrapped in the nitrocellulose filter detection layers
There are detection line and nature controlling line.
In a kind of embodiment, the support plate is PVC bottom plate.
In a kind of embodiment, in the nitrocellulose filter detection layers, the detection line is located at from sample-adding end nearlyr one
Side, nature controlling line are located at from sample-adding end side farther out.
In a kind of embodiment, campylobacter jejuni monoclonal antibody II is coated in the detection line.
In a kind of embodiment, sheep anti-mouse igg is coated on the nature controlling line.
In a kind of embodiment, the detection target of the campylobacter jejuni monoclonal antibody I is campylobacter jejuni CjaA egg
It is white, that is to say, that campylobacter jejuni monoclonal antibody I is the monoclonal antibody for campylobacter jejuni CjaA albumen.
In a kind of embodiment, it is campylobacter jejuni CjaA egg that the campylobacter jejuni monoclonal antibody II, which detects target,
It is white, that is to say, that campylobacter jejuni monoclonal antibody II is the monoclonal antibody for campylobacter jejuni CjaA albumen.
In a kind of embodiment, the campylobacter jejuni monoclonal antibody I and the campylobacter jejuni monoclonal antibody II
The epitope of the detection target identified is different.
In a kind of embodiment, the campylobacter jejuni monoclonal antibody I includes light chain and heavy chain, and the light chain includes ammonia
Base acid sequence CDR region as shown in NO.1~3 SEQ ID, the heavy chain include the amino acid sequence such as institute of SEQ ID NO.5~7
The CDR region shown.
In a kind of embodiment, the heavy chain is connected with light chain by disulfide bond.
In a kind of embodiment, the campylobacter jejuni monoclonal antibody I, the amino acid sequence of light chain variable region is
SEQ ID NO.4 or its conservative series of variation, the amino acid sequence of heavy chain variable region are SEQ ID NO.8 or its conservative
Series of variation.
In a kind of embodiment, the coding nucleotide sequence of the light chain variable region is SEQ ID NO.12 or its conservative
Series of variation, the coding nucleotide sequence of the heavy chain variable region are SEQ ID NO.16 or its conservative series of variation.
In a kind of embodiment, the campylobacter jejuni monoclonal antibody II includes light chain and heavy chain, and the light chain includes
Amino acid sequence CDR region as shown in NO.17~19 SEQ ID, the heavy chain include amino acid sequence such as SEQ ID NO.21
CDR region shown in~23.
In a kind of embodiment, the heavy chain is connected with light chain by disulfide bond.
In a kind of embodiment, the campylobacter jejuni monoclonal antibody II, the amino acid sequence of light chain variable region is
SEQ ID NO.20 or its conservative series of variation, the amino acid sequence of heavy chain variable region is SEQ ID NO.24 or it is conservative
Form variation sequence.
In a kind of embodiment, the coding nucleotide sequence of the light chain variable region is SEQ ID NO.28 or its conservative
Series of variation, the coding nucleotide sequence of the heavy chain variable region are SEQ ID NO.32 or its conservative series of variation.
In a kind of embodiment, the sample pad selects glass fibre element film.
In a kind of embodiment, the gold-labelled pad selects glass fibre element film.
The second aspect of the present invention provides the preparation method for being previously used for the gold label test strip of campylobacter jejuni detection, including
Following steps:
1) gold-labelled pad is sprayed with the campylobacter jejuni monoclonal antibody I of colloid gold label, be made comprising colloid gold label
The gold-labelled pad of campylobacter jejuni monoclonal antibody I；
2) campylobacter jejuni monoclonal antibody is sprayed respectively in the detection line of nitrocellulose filter detection layers and nature controlling line
II and sheep anti-mouse igg, the nitrocellulose filter after coating is made；
3) successively by sample pad, step 1) preparation gold-labelled pad, the nitrocellulose filter detection layers of step 2) preparation, water absorption pad
It is pasted on support plate, test strip is made in cutting.
In a kind of embodiment, the detection target of the campylobacter jejuni monoclonal antibody I is campylobacter jejuni CjaA egg
It is white, that is to say, that campylobacter jejuni monoclonal antibody I is the monoclonal antibody for campylobacter jejuni CjaA albumen.
In a kind of embodiment, it is campylobacter jejuni CjaA egg that the campylobacter jejuni monoclonal antibody II, which detects target,
It is white, that is to say, that campylobacter jejuni monoclonal antibody II is the monoclonal antibody for campylobacter jejuni CjaA albumen.
In a kind of embodiment, the campylobacter jejuni monoclonal antibody I and the campylobacter jejuni monoclonal antibody II
The epitope of the detection target identified is different.
In a kind of embodiment, the sample pad selects glass fibre element film.
In a kind of embodiment, the gold-labelled pad selects glass fibre element film.
Third aspect present invention provides the purposes that aforementioned gold label test strip is used to prepare campylobacter jejuni testing product.
The fourth aspect of the present invention provides a kind of campylobacter jejuni detection kit, including is previously used for campylobacter jejuni
It the gold label test strip of detection and gets stuck, described to get stuck including back card and upper cover, the back card is equipped with test paper clamp bar slot, is previously used for
For the gold label test strip of campylobacter jejuni detection in the test paper clamp bar slot, the upper cover is equipped with testing window and well, institute
The position for stating testing window is matched with the position of the detection line and nature controlling line, the position of the well and the sample pad
Position matches.
In a kind of embodiment, described get stuck is got stuck for plastics.
Compared with prior art, the invention has the following beneficial effects:
(1) easy to be quick, detection process only needs 10 minutes, the quick detection suitable for batch samples；
(2) high specificity, high sensitivity, the concentration limit of campylobacter jejuni are 100CFU/ μ L；
(3) easy to carry, direct loading is not necessarily to Sample pretreatment, and required detection sample size is few, is suitble to on-site test；
(4) accuracy in detection is high, can be used as the substitution detection scheme or complementarity method of campylobacter jejuni bacterial cultivation；
(5) application value is high, and commercial promise is good, quickly detects for production of poultry meat processing site campylobacter jejuni and provides technology
Support.
Detailed description of the invention
Fig. 1: the structural schematic diagram of the gold label test strip for campylobacter jejuni detection of the invention, wherein 1 is support plate
(can be PVC bottom plate), 2 be sample pad, 3 be gold-labelled pad, 4 be nitrocellulose filter detection layers, 5 be detection line, 6 be Quality Control
Line, 7 are water absorption pad.
Fig. 2: the sensitivity test result figure of the gold label test strip for campylobacter jejuni detection of the invention.
Fig. 3: result figure is analyzed in the cross reaction of the gold label test strip of campylobacter jejuni detection of the invention.
Fig. 4: the stability test result figure of the gold label test strip of campylobacter jejuni detection of the invention.
Specific embodiment
Colloidal gold immunochromatographimethod reaction (Gold immunochromatography assay, GICA) is a kind of emerging
Quick diagnosis technology based on antigen-antibody immune response, colloid gold label, protein chromatographic, have it is easy quickly, result it is visual,
Without complicated operation skill, it is easy to carry the advantages that, oneself becomes the research hotspot in in-vitro diagnosis field, is applicable in and examines on site
It surveys.As the immuno analytical method that a kind of film relies on, GICA test strips have been widely used in the detection of small-molecule substance, such as
Pesticide residue, progestational hormone, the antibody etc. in serum.However in the detection field of macromolecular substances, test strips research and development difficulty is heavy,
The especially detection of bacterium.The volume of bacterium is larger, and climbing for chromatographic film is fast limited with albumen bearing capacity, this is to a certain extent
Effective combination of antigen-antibody is affected, and limits the sensibility of system.Along with the condition of culture of campylobacter jejuni harshness,
Most of campylobacteriosis all have self limiting.CjaA albumen belongs to campylobacter jejuni ABC type cysteine movement system, big portion
Amino acid is divided to be exposed to outer membrane, present invention discover that CjaA protein-specific is good, conservative is high, homology is high, can be used as jejunum
The detection target of Campylobacter spp.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
1, the preparation and acquisition of campylobacter jejuni monoclonal antibody I and campylobacter jejuni monoclonal antibody II
The detection target of campylobacter jejuni monoclonal antibody I in the present invention is campylobacter jejuni CjaA albumen, that is,
Say that campylobacter jejuni monoclonal antibody I is the monoclonal antibody for campylobacter jejuni CjaA albumen.Jejunum in the present invention is curved
It is also campylobacter jejuni CjaA albumen that aspergillus monoclonal antibody II, which detects target, that is to say, that campylobacter jejuni monoclonal antibody II
It is another monoclonal antibody for campylobacter jejuni CjaA albumen.Campylobacter jejuni monoclonal antibody I and campylobacter jejuni are single
The epitope for the detection target that clonal antibody II is identified is different.
The present invention obtains campylobacter jejuni monoclonal antibody I by hybridoma cell strain screening technique and campylobacter jejuni is single
Clonal antibody II send company to carry out sequencing identification.The result shows that:
The amino acid sequence such as SEQ of the light chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.1, specifically:
RASQSISDYLH。
The amino acid sequence such as SEQ of the light chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.2, specifically:
YASQSIS。
The amino acid sequence such as SEQ of the light chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.3, specifically:
QNGHSFPLT。
The amino acid sequence of the light chain variable region of campylobacter jejuni monoclonal antibody I is as shown in SEQ ID NO.4, specifically
Are as follows:
MVSTSQLLGLLLFWTSASRCDIVMTQSPATLSVTPGDSVSLSCRASQSISDYLHWYQQKSHGSPRLLI
KYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK。
That is, the light chain variable region of campylobacter jejuni monoclonal antibody I contains 127 amino acid.
The amino acid sequence such as SEQ of the heavy chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.5, specifically:
SYVIF。
The amino acid sequence such as SEQ of the heavy chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.6, specifically:
YINPYNDGTKYDENFKG。
The amino acid sequence such as SEQ of the heavy chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.7, specifically:
SIMITRGWYFDV。
The amino acid sequence of the heavy chain variable region of campylobacter jejuni monoclonal antibody I as shown in SEQ ID NO., specifically:
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGTSVKMSCKASGYTFISYVIFWVKQKPGQGLEWIG
YINPYNDGTKYDENFKGKATLTSDKSSSAAYMELSSLTSEDSAVYYCARSIMITRGWYFDVWGAGTTVTVSS。
That is, the heavy chain variable region of campylobacter jejuni monoclonal antibody I contains 140 amino acid.
Accordingly, the nucleotide sequence of the light chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody I
As shown in SEQ ID NO.9, specifically:
AGGGCCAGCCAGTCTATTAGCGACTACTTACAC。
The nucleotide sequence such as SEQ of the light chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.10, specifically:
TATGCTTCCCAATCCATCTCT。
The nucleotide sequence such as SEQ of the light chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.11, specifically:
CAAAATGGTCACAGCTTTCCGCTCACG。
The nucleotide sequence of the light chain variable region of campylobacter jejuni monoclonal antibody I is as shown in SEQ ID NO.12, specifically
Are as follows:
ATGGTGTCCACTTCTCAGCTCCTTGGACTTTTGCTTTTCTGGACTTCAGCCTCCAGATGTGACATTGT
GATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCTCTCTTTCCTGCAGGGCCAGCCAGTCT
ATTAGCGACTACTTACACTGGTATCAACAAAAATCACATGGGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAAT
CCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGA
ACCTGAAGATGTTGGAGTGTATTACTGTCAAAATGGTCACAGCTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTG
GAGCTGAAA。
That is, the nucleotide sequence of the light chain variable region of campylobacter jejuni monoclonal antibody I contains 381 bases.
The nucleotide sequence such as SEQ of the heavy chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.13, specifically:
AGCTATGTTATCTTT。
The nucleotide sequence such as SEQ of the heavy chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.14, specifically:
TATATTAATCCTTACAATGATGGTACTAAATACGATGAGAACTTCAAAGGC。
The nucleotide sequence such as SEQ of the heavy chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody I
Shown in ID NO.15, specifically:
TCGATTATGATTACGAGGGGCTGGTACTTCGATGTC。
The nucleotide sequence of the heavy chain variable region of campylobacter jejuni monoclonal antibody I is as shown in SEQ ID NO.16, specifically
Are as follows:
ATGGAATGGAGTTGGATATTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCT
GCAGCAGTCTGGACCTGAACTGGTAAAGCCTGGGACTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTC
ATTAGCTATGTTATCTTTTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACA
ATGATGGTACTAAATACGATGAGAACTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCGCAGCCTA
CATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGATTATGATTACGAGGGGC
TGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA。
That is, the nucleotide sequence of the heavy chain variable region of campylobacter jejuni monoclonal antibody I contains 420 bases.
The amino acid sequence such as SEQ of the light chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.17, specifically:
KASDHINKWLA。
The amino acid sequence such as SEQ of the light chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.18, specifically:
SATSLET。
The amino acid sequence such as SEQ of the light chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.19, specifically:
QQYWSTPWT。
The amino acid sequence of the light chain variable region of campylobacter jejuni monoclonal antibody II is as shown in SEQ ID NO.20, specifically
Are as follows:
MKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVTITCKASDHINKWLAWYQQKPGNAPRLLI
SSATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK。
That is, the light chain variable region of campylobacter jejuni monoclonal antibody II contains 127 amino acid.
The amino acid sequence such as SEQ of the heavy chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.21, specifically:
SDVIH。
The amino acid sequence such as SEQ of the heavy chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.22, specifically:
YFNPYNDVTNYNENFKG。
The amino acid sequence such as SEQ of the heavy chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.23, specifically:
SGHALDY。
The amino acid sequence of the heavy chain variable region of campylobacter jejuni monoclonal antibody II is as shown in SEQ ID NO.24, specifically
Are as follows:
MEWSWIFLFLLSGTAGVYSEVQLQQSGPELVKPGASMKMSCKASGYIFTSDVIHWVRQKPGQGLEWIG
YFNPYNDVTNYNENFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSGHALDYWGQGTSVTVSS。
That is, the heavy chain variable region of campylobacter jejuni monoclonal antibody II contains 135 amino acid.
Accordingly, the nucleotides sequence of the light chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody II
It arranges as shown in SEQ ID NO.25, specifically:
AAGGCAAGTGACCACATTAATAAATGGTTAGCC。
The nucleotide sequence such as SEQ of the light chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.26, specifically:
AGTGCAACCAGTTTGGAAACT。
The nucleotide sequence such as SEQ of the light chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.27, specifically:
CAACAGTATTGGAGTACTCCGTGGACG。
The nucleotide sequence of the light chain variable region of campylobacter jejuni monoclonal antibody II is as shown in SEQ ID NO.28, specifically
Are as follows:
ATGAAGTTTCCTTCTCAACTTCTGCTCTTCCTGCTGTTCAGAATCACAGGCATAATATGTGACATCCA
GATGACACAATCTTCATCCTACTTGTCTGTGTCTCTAGGAGGCAGAGTCACCATTACTTGCAAGGCAAGTGACCAC
ATTAATAAATGGTTAGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTAGTGCAACCAGTT
TGGAAACTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCA
GACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTG
GAAATCAAA。
That is, the nucleotide sequence of the light chain variable region of campylobacter jejuni monoclonal antibody II contains 381 bases.
The nucleotide sequence such as SEQ of the heavy chain variable region complementary determining region 1 (CDR1) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.29, specifically:
AGCGATGTTATTCAC。
The nucleotide sequence such as SEQ of the heavy chain variable region complementary determining region 2 (CDR2) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.30, specifically:
TATTTTAATCCTTACAATGATGTTACTAACTACAATGAGAACTTCAAAGGC。
The nucleotide sequence such as SEQ of the heavy chain variable region complementary determining region 3 (CDR3) of campylobacter jejuni monoclonal antibody II
Shown in ID NO.31, specifically:
AGCGGACATGCTTTGGACTAC。
The nucleotide sequence of the heavy chain variable region of campylobacter jejuni monoclonal antibody II is as shown in SEQ ID NO.32, specifically
Are as follows:
ATGGAATGGAGTTGGATATTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCTACTCTGAGGTCCAGCT
GCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAATGAAGATGTCCTGCAAGGCTTCTGGATACATATTC
ACTAGCGATGTTATTCACTGGGTGAGGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACA
ATGATGTTACTAACTACAATGAGAACTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTA
CATGGAGCTCAGCAGCCTGACCTCTGAAGACTCTGCGGTCTATTACTGTGCAAGAAGCGGACATGCTTTGGACTAC
TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。
That is, the nucleotide sequence of the heavy chain variable region of campylobacter jejuni monoclonal antibody II contains 405 bases.
Those skilled in the art are knowing campylobacter jejuni monoclonal antibody I and campylobacter jejuni monoclonal antibody II
After light chain and sequence of heavy chain, campylobacter jejuni monoclonal antibody I is prepared by the way that conventional technique for gene engineering is i.e. repeatable
With campylobacter jejuni monoclonal antibody II.
For example, the present invention is repeated using following genetic engineering, to prepare campylobacter jejuni monoclonal antibody I curved with jejunum
Aspergillus monoclonal antibody II:
(1) the antibody variable region coded sequence that sequencing obtains expression will be inserted into Seamless Cloning method to carry
In body, recombinant expression carrier is obtained；
(2) recombinant expression carrier of acquisition is converted into host cell；
(3) host cell under conditions of being suitble to monoclonal antibody expression after conversion obtained by incubation step (2)；
(4) acquisition monoclonal antibody is isolated and purified.
Sequencing identification is carried out to the campylobacter jejuni monoclonal antibody I and campylobacter jejuni monoclonal antibody II of acquisition,
Expression is correct, sequence and expected consistent.
2, the preparation of colloidal gold solution and gold-labelled monoclonal antibody compound
(1) preparation of colloidal gold
Colloidal gold is the prior art, and those skilled in the art can be obtained by commercially available approach, can also voluntarily be prepared.From
It, can be by the following method when row preparation:
Au colloidal nanoparticles are prepared using sodium citrate.200mL deionized water and 2mL matter are added in conical flask
The chlorauric acid solution that concentration is 1% is measured, is heated to boiling under stirring well.Rapidly joining 3mL mass concentration is 1%
Trisodium citrate.Chlorauric acid solution successively becomes black from light green color, after eventually becoming red, continues heating 10-15 minutes, so
It cools down at room temperature afterwards, 4 DEG C are kept in dark place for use.
(2) the campylobacter jejuni monoclonal antibody I of colloid gold label
The colloidal gold solution 10mL prepared is taken, with the K of 0.1M2CO3PH value is adjusted to 8.0, it is empty that 265 μ g are added dropwise in stirring
Intestines Campylobacter spp monoclonal antibody I continues stirring 1 hour.0.5mL10%BSA solution is added dropwise, is stirred to react 0.5 hour, then drips
Add 0.2mL 10%PEG-20000 solution, is stood overnight after being stirred to react 45 minutes in 4 DEG C.Finally by 4 DEG C of solution, 1,
500rpm is centrifuged 15 minutes, abandons precipitating.It by supernatant in 4 DEG C, 10,000rpm, is centrifuged 45 minutes, abandons supernatant and with 1mL 0.01M
Precipitating is resuspended in PB solution (containing 1%BSA and 0.1%Triton X-100), obtains the campylobacter jejuni Dan Ke of colloid gold label
Grand antibody I.
2, the combination of the spraying of antibody and test strips
As shown in Figure 1, the gold label test strip for campylobacter jejuni detection of the invention includes that support plate 1 (can be PVC
Bottom plate) and positioned at the sample pad 2 being arranged successively from sample-adding end, gold-labelled pad 3, nitrocellulose filter detection for supporting plate surface
Layer 4 and water absorption pad 7 includes the campylobacter jejuni monoclonal antibody I of colloid gold label, the nitrocellulose in the gold-labelled pad
Detection line 5 and nature controlling line 6 are coated in film detection layers 4.In the nitrocellulose filter detection layers 4, the detection line 5 be located at from
It is loaded the nearlyr side in end, nature controlling line 6 is located at from sample-adding end side farther out.Campylobacter jejuni Dan Ke is coated in the detection line 5
Grand antibody II.Sheep anti-mouse igg is coated on the nature controlling line 6.
Test strips of the invention use colloidal gold immunochromatographimethod technology, select campylobacter jejuni monoclonal antibody I as solid
Phase object, using whether containing tested bacteria in double antibody sandwich method test sample.When containing campylobacter jejuni in measuring samples,
Antigen first acts on forward movement in conjunction with the campylobacter jejuni monoclonal antibody I of colloid gold label, and through chromatography, examines when encountering
When the campylobacter jejuni monoclonal antibody II of the identifying purpose albumen another kind epitope on survey line, it is single to form campylobacter jejuni
II-antigen of clonal antibody-colloid gold label campylobacter jejuni monoclonal antibody I, it is red heavy to be formed after being enriched in detection line
Shallow lake line.Without and antigen binding colloid gold label campylobacter jejuni monoclonal antibody I and nature controlling line on the sheep anti mouse that embeds
IgG antibody combines, and forms the campylobacter jejuni monoclonal antibody I- secondary antibody compound of colloid gold label, is enriched on nature controlling line,
Form red precipitate line.
When preparation,
Three-dimensional specking film gold spraying instrument is debugged, the campylobacter jejuni monoclonal antibody I of the colloid gold label of acquisition is uniformly sprayed
It being applied on glass fibre element film, spouting liquid is 30 μ L/cm, drying at room temperature, gold-labelled pad is obtained, envelope is spare.By campylobacter jejuni
Monoclonal antibody II (2mg/mL) and sheep anti-mouse igg (1.0mg/mL) are equably sprayed on nitrocellulose filter respectively, spouting liquid
For 0.7 μ L/cm to get arrive detection line and nature controlling line.By the nitrocellulose filter of spray coated antibody, that is, nitrocellulose filter
Detection layers are immersed in 1%BSA solution, 37 DEG C water-bath 2 hours, drying at room temperature, seal it is spare.By PVC bottom plate, nitrocellulose
Film detection layers, sample pad, gold-labelled pad, detection line, nature controlling line and water absorption pad combination are to get colloidal gold immuno-chromatography test paper strip, step
It is rapid briefly as follows: nitrocellulose filter detection layers being first sticked to the middle position of PVC bottom plate, then paste the front end of gold-labelled pad
It is pressed on nitrocellulose filter detection layers rear end at about 2mm, then pastes sample and be padded on PVC bottom plate least significant end and push down gold-labelled pad
Water absorption pad is finally pasted onto the front end of PVC bottom plate and its end is made to be pressed in the front ends of nitrocellulose filter detection layers by rear end,
It obtains and (can be also simply referred to as colloidal gold strip or the curved bacterium colloid gold immune of jejunum for the gold label test strip of campylobacter jejuni detection
Chromatograph test strip or colloidal gold strip).Test strips are cut into the specification of 3.8mm × 60mm, are sealed spare.
3, sensitivity test
The detection method and result judgement of gold label test strip for campylobacter jejuni detection of the invention are according to as follows: will
Sample pad is added in testing sample solution, observes the colour developing situation of detection line and nature controlling line after ten minutes.If detection line and Quality Control
Line all shows that red illustrates there is campylobacter jejuni in sample；If redfree band at detection line and shown at nature controlling line red
Color then illustrates do not have campylobacter jejuni in sample；If band does not occur in nature controlling line, no matter whether detection line there are red stripes,
All represent the failure of test strips system.
It has been prepared by the test detection of the sample-adding of campylobacter jejuni and non-campylobacter jejuni (Tables 1 and 2) bacteria suspension
For campylobacter jejuni detection gold label test strip specificity.By 60 μ L testing sample solutions be added sample pad, 10 minutes
The colour developing situation of detection line and nature controlling line is observed afterwards.Experiment is repeated 3 times.
The campylobacter jejuni type strain NCTC11168 of recovery cryopreservation prepares bacterial suspension.10 times of bacteria suspension are incremented by
It is diluted to 1.0 × 106CFU/μL-1.0×101This 6 concentration of CFU/ μ L carry out sample-adding test respectively, and blank control group is arranged,
The campylobacter jejuni Monitoring lower-cut of test strips is investigated, experiment is repeated 3 times.The results show that the sky of colloidal gold strip of the invention
Intestines Campylobacter spp concentration limit is 100CFU/ μ L (Fig. 2).
4, cross reaction is analyzed
Glue of the invention is detected by the sample-adding test of campylobacter jejuni and non-campylobacter jejuni (Tables 1 and 2) bacteria suspension
The specificity of body gold test paper strip.Sample pad is added in 60 μ L testing sample solutions, observes detection line and nature controlling line after ten minutes
Develop the color situation.Experiment is repeated 3 times.
Campylobacter jejuni used in the experiment of table 1
Non- campylobacter jejuni used in the experiment of table 2
aNCTC, Chinese medicine Microbiological Culture Collection administrative center；
bCCTCC, Chinese Industrial Standards (CIS) strain collections；
cATCC, Unite States Standard strain collections；
The results show that the specificity of colloidal gold strip detection campylobacter jejuni of the invention is good.96 plants of jejunum campylobacters
The bacteria suspension testing result of bacterium (including type strain and separation strains) is the positive, test strips and other 50 plants of Campylobacter Colis, 1 plant
The bacterium no cross reaction (Fig. 3) of campylobacter fetus and 9 plants of un-flexed Pseudomonas.
5, stability test
2 experimental groups are arranged in stability test altogether: stability test group and batch internal stability test group between batch.Each test
Group includes 5 positive samples and 5 negative samples.Colloidal gold strip between batch in stability test group is different batches system
Standby test strips, the test strips of batch internal stability test group are the colloidal gold strip of same batch preparation.React same time
Afterwards, the colour developing situation of detection line and nature controlling line is observed.Experiment is repeated 3 times.The results show that colloidal gold strip has good stability
(Fig. 4).
Embodiment 2
Using the campylobacter jejuni in the curved bacterium colloidal gold immuno-chromatography test paper strip test sample of jejunum of the invention.
1. sample analog detection
After harvesting bacterium, 10 times of bacterial suspension are incremented by by the campylobacter jejuni type strain NCTC11168 of recovery cryopreservation
It is diluted to 1.0 × 106CFU/μL-1.0×101This 6 concentration of CFU/ μ L, and blank control group is set.Take the bacterium of each concentration
Suspension 1mL is separately added into the excrement sample of 1g chicken, and analog sample is used as after mixing.Draw each 60 μ L of simulation bacterium solution of each concentration
Test strips sample-adding test is carried out, while using the testing result of bacterial cultivation as control.Experimental result shows, glue of the invention
The campylobacter jejuni concentration limit of body gold test paper strip is 100CFU/ μ L, the detection sensitivity and jejunum campylobacter of analog sample
The detection sensitivity of bacterium pure culture is consistent, and the campylobacter jejuni positive analog sample that test strips detect, bacterial cultivation
Also test positive.
2. the field quick detection of poultry surface wipes sample jejuni
In production of poultry meat processing site, 233 parts of pork surface wipes sample is acquired, is soaked using PBS (sterilizing, pH7.2)
Cotton balls wipes poultry surface, is subsequently placed into sterile plastics sampler bag.It is curved using the jejunum in test strips on-site test sample
When aspergillus, the PBS in cotton balls sample is squeezed, draws the sample pad that GICA test strips are added in 60 μ L liquid.Test paper is observed after ten minutes
The testing result of item.Then the sack of sampler bag is tightened, transports laboratory back at once, and adopt by the detection of conventional bacteria cultivation
Campylobacter jejuni in sample bag in remaining sample.
Conventional bacteria cultivation and colloidal gold strip method detect 35 parts of campylobacter jejuni positive sample (table 3) altogether, to pass
The testing result of system bacterial cultivation is standard, and the accuracy that test strips detect campylobacter jejuni is 93%, be can be applied to large quantities of
Measure the field quick detection of poultry sample jejuni.
3 colloidal gold strip of table detects poultry surface wipes sample jejuni
In conclusion colloidal gold immuno-chromatography test paper strip provided by the present invention, it is hollow to can be used for poultry surface wipes sample
Intestines Campylobacter spp field quick detection.Detection method of the invention using the monoclonal antibody of two kinds of different epitopes of campylobacter jejuni as
Gold conjugated antibody and detection antibody are used for quickly detecting campylobacter jejuni.The test strips have portable quick, operation letter
It is single, not by the interference of environmental condition, the advantages that specificity is good, high sensitivity, entire detection process only needs 10 minutes.The present invention is suitable
Technical support is provided for units such as poultry meat processing enterprise, inspection and quarantine, and for the real-time extensive detection of campylobacter jejuni.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Sequence table
<110>Yangzhou University
<120>a kind of gold label test strip and its preparation and application for campylobacter jejuni detection
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atggaatgga gttggatatt tctctttctc ctgtcaggaa ctgcaggtgt ccactctgag 60
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tgtaaggctt ctggatacac attcattagc tatgttatct tttgggtgaa gcagaagcct 180
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Lys Ala Ser Asp His Ile Asn Lys Trp Leu Ala
1 5 10
<210> 18
<211> 7
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Ser Ala Thr Ser Leu Glu Thr
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Ser Gly His Ala Leu Asp Tyr
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aaggcaagtg accacattaa taaatggtta gcc 33
<210> 26
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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caacagtatt ggagtactcc gtggacg 27
<210> 28
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<213>artificial sequence (Artificial Sequence)
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attacttgca aggcaagtga ccacattaat aaatggttag cctggtatca gcagaaacca 180
ggaaatgctc ctaggctctt aatatctagt gcaaccagtt tggaaactgg ggttccttca 240
agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 300
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<210> 29
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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agcgatgtta ttcac 15
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<213>artificial sequence (Artificial Sequence)
<400> 30
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<210> 31
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
agcggacatg ctttggacta c 21
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<211> 405
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<213>artificial sequence (Artificial Sequence)
<400> 32
atggaatgga gttggatatt tctctttctc ctgtcaggaa ctgcaggtgt ctactctgag 60
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tgcaaggctt ctggatacat attcactagc gatgttattc actgggtgag gcagaagcct 180
gggcagggcc ttgagtggat tggatatttt aatccttaca atgatgttac taactacaat 240
gagaacttca aaggcaaggc cacactgact tcagacaaat cctccagcac agcctacatg 300
gagctcagca gcctgacctc tgaagactct gcggtctatt actgtgcaag aagcggacat 360
gctttggact actggggtca aggaacctca gtcaccgtct cctca 405
Claims (12)
1. a kind of gold label test strip for campylobacter jejuni detection, including support plate and positioned at support plate surface from adding
Sample pad, gold-labelled pad, nitrocellulose filter detection layers and the water absorption pad that sample end is arranged successively include colloidal gold in the gold-labelled pad
The campylobacter jejuni monoclonal antibody I of label is coated with detection line and nature controlling line in the nitrocellulose filter detection layers.
2. gold label test strip according to claim 1, which is characterized in that the inspection of the campylobacter jejuni monoclonal antibody I
Survey target is campylobacter jejuni CjaA albumen.
3. gold label test strip according to claim 1, which is characterized in that be coated with campylobacter jejuni list in the detection line
Clonal antibody II.
4. gold label test strip according to claim 3, which is characterized in that the campylobacter jejuni monoclonal antibody II detects
Target is campylobacter jejuni CjaA albumen.
5. gold label test strip according to claim 3, which is characterized in that the campylobacter jejuni monoclonal antibody II and institute
The epitope for stating the detection target that campylobacter jejuni monoclonal antibody I is identified is different.
6. gold label test strip according to claim 3, which is characterized in that the campylobacter jejuni monoclonal antibody I includes
Light chain and heavy chain, the light chain include amino acid sequence CDR region as shown in NO.1~3 SEQ ID, and the heavy chain includes amino
Acid sequence CDR region as shown in NO.5~7 SEQ ID.
7. gold label test strip according to claim 3, which is characterized in that the campylobacter jejuni monoclonal antibody II includes
Light chain and heavy chain, the light chain include amino acid sequence CDR region as shown in NO.17~19 SEQ ID, and the heavy chain includes ammonia
Base acid sequence CDR region as shown in NO.21~23 SEQ ID.
8. gold label test strip according to claim 1, which is characterized in that be coated with sheep anti-mouse igg on the nature controlling line.
9. gold label test strip according to claim 1, which is characterized in that further include any one of following characteristics or more
:
(1) sample pad selects glass fibre element film；(2) gold-labelled pad selects glass fibre element film；(3) support plate
For PVC bottom plate；(4) in the nitrocellulose filter detection layers, the detection line is located at side closer from sample-adding end, nature controlling line position
In from sample-adding end side farther out.
10. the preparation method of gold label test strip as described in any one of claims 1 to 9, which is characterized in that including walking as follows
It is rapid:
1) gold-labelled pad is sprayed with the campylobacter jejuni monoclonal antibody I of colloid gold label, the jejunum comprising colloid gold label is made
The gold-labelled pad of Campylobacter spp monoclonal antibody I；
2) sprayed respectively in the detection line of nitrocellulose filter detection layers and nature controlling line campylobacter jejuni monoclonal antibody II and
Sheep anti-mouse igg, the nitrocellulose filter after coating is made；
3) sample pad, step 1) preparation gold-labelled pad, the nitrocellulose filter detection layers of step 2) preparation, water absorption pad are successively pasted
On support plate, test strip is made in cutting.
11. the purposes that gold label test strip as described in any one of claims 1 to 9 is used to prepare campylobacter jejuni testing product.
12. a kind of campylobacter jejuni detection kit is detected including any campylobacter jejuni that is used for of claim 1~9
Gold label test strip and get stuck, it is described to get stuck including back card and upper cover, the back card be equipped with test paper clamp bar slot, such as claim 1
In the test paper clamp bar slot, the upper cover is equipped with gold label test strip described in~9 any one for campylobacter jejuni detection
Testing window and well, the position of the testing window are matched with the position of the detection line and nature controlling line, the well
Position is matched with the position of the sample pad.
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| CN201811008658.1A CN109142726A (en) | 2018-08-29 | 2018-08-29 | A kind of gold label test strip and its preparation and application for campylobacter jejuni detection |
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| CN201811008658.1A Pending CN109142726A (en) | 2018-08-29 | 2018-08-29 | A kind of gold label test strip and its preparation and application for campylobacter jejuni detection |
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| CN (1) | CN109142726A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020039A (en) * | 2019-12-30 | 2020-04-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Campylobacter jejuni species specific molecular target and rapid detection method thereof |
| CN111440230A (en) * | 2020-04-03 | 2020-07-24 | 金华职业技术学院 | Construction and application of tandem epitope polypeptide, gene, recombinant plasmid and recombinant bacteria of campylobacter jejuni outer membrane protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792992A (en) * | 2015-05-04 | 2015-07-22 | 潍坊市康华生物技术有限公司 | Colloidal gold for campylobacter jejuni antigen detection, glass fiber sample pad containing colloidal gold, detection reagent strip and detection kit |
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Patent Citations (1)
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| CN104792992A (en) * | 2015-05-04 | 2015-07-22 | 潍坊市康华生物技术有限公司 | Colloidal gold for campylobacter jejuni antigen detection, glass fiber sample pad containing colloidal gold, detection reagent strip and detection kit |
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| 尹衍新等: "基于CjaA蛋白检测鸡空肠弯曲菌抗体的间接ELISA方法建立及其单克隆抗体的研制", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020039A (en) * | 2019-12-30 | 2020-04-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Campylobacter jejuni species specific molecular target and rapid detection method thereof |
| CN111440230A (en) * | 2020-04-03 | 2020-07-24 | 金华职业技术学院 | Construction and application of tandem epitope polypeptide, gene, recombinant plasmid and recombinant bacteria of campylobacter jejuni outer membrane protein |
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Application publication date: 20190104 |