CN204287202U - A kind of Mycoplasma bovis test strip - Google Patents

Abstract

The utility model discloses a kind of Mycoplasma bovis antibody test test strips.Test strips of the present utility model by base plate, be arranged on base plate that linearly aligned absorption of sample pad, the colloidal gold pad being coated with antigen, nitrocellulose membrane and adsorptive pads form successively, nitrocellulose filter is coated with nature controlling line and detection line, wherein detection line bag quilt is goat anti-ox two anti-igg, and nature controlling line is many anti-igg of rabbit source P48 fusion.Test strips of the present utility model can detect Mycoplasma bovis antibody, comprise serum antibody and whey antibody, and it is low to have cost, fast simple, convenient, and without the need to specific installation and instrument, detection sensitivity is high, high specificity, the advantages such as applied range.

Description

A kind of Mycoplasma bovis test strip

Technical field

The utility model relates to a kind of biological detection test strips, and the utility model relates to a kind of Mycoplasma bovis antibody test test strips exactly.

Background technology

Mycoplasma bovis is a kind of pathogenic pathogen seriously, cattle respiratory disease, mastadenitis of cow, arthritis, keratoconjunctivitis, genital inflammation and miscarriage and the various diseases such as infertile can be caused, and once infect the scabies secondary infection that can cause other cause of disease, such as suppurative Pyrogenes, pasteurella multocida, Mannheimia haemolytica, lethargic sleep haemophilus, bovine parainfluenza virus 3 type, herpesviral 1 type, Bovine Respiratory Syncytial virus, bovine viral diarrhea virus and bovine coronavirus etc.The Mycoplasma bovis incidence of disease is 50%-100%, and case fatality rate, up to 10%-50%, causes huge economic loss to China's cattle-raising.This disease China actual popularity and epidemiology information is still comparatively deficient accurately, cause the reason of this phenomenon, mainly China lacks the correlation technique that effective Mycoplasma bovis detects at present.

The technology of the detection Mycoplasma bovis of current existence has: 1. Antigen isolation and identification, but Mycoplasma bovis is separated and is often subject to the antibiotic impact of clinical practice, separation difficulty, and separation nutrient culture media nutritional requirement is high, isolate identification difficulty is large, sensitivity is low, the method that can not infect as Rapid&Early diagnosis Mycoplasma bovis, also be difficult to as common detection methods penetration and promotion (coming from Caswell J L, Archambault M. Mycoplasma bovis pneumonia in cattle), 2.PCR detection method (comes from Thomas A, Dizier I, Linden A, et al .Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis), this detection method needs to carry out more complicated process (grinding to samples, centrifugal, extract DNA etc.), though detection method is sensitive, but easily there is false positive because polluting and dealing with improperly than being easier to, affect the accuracy of result of determination, in addition certain laboratory condition and instrument and equipment is also needed, and higher cost, be not suitable for clinical or basic unit's use.3. indirect ELISA applies topmost Serology test at present, successively useful whole bacterial protein bag quilt and expressing protein (come from Grand D L as the method for envelope antigen in the world, Calavas D, Brank M, et al. Serological prevalence of Mycoplasma bovis infection in suckling beef cattle in France), the method specificity deficiency of whole bacterial protein is now seldom applied, expressing protein antigen ELISA method is existing commercially produced product at present, but its which antigen protein used is not announced.In addition, ELISA method is only adapted at laboratory and detects, and operation steps relative complex needs professional to operate, and needs certain laboratory condition and instrument and equipment to carry out determination and analysis result.4. utilize the indirect hemagglutination detection method that P48 recombinant expression protein bag is set up by sheep red blood cell (SRBC), operation is relatively simple, but also need 2 ~ 3 hours just can result of determination, and sensitivity is weak, needs specific constant incubator.

Summary of the invention

The utility model provides one can overcome prior art deficiency, the detection Mycoplasma bovis serum antibody that energy is simple and direct, quick, sensitive, the test strip of whey antibody.

A kind of test strips detecting Mycoplasma bovis antibody of the present utility model by base plate, be arranged on base plate that linearly aligned absorption of sample pad, the colloidal gold pad being coated with antigen, nitrocellulose membrane and adsorptive pads form successively, nitrocellulose filter is coated with nature controlling line and detection line, the antigen of the colloidal gold pad bag quilt in the utility model test strips is Mycoplasma bovis P48 fusion, detection line bag quilt be goat anti-ox two anti-igg, nature controlling line is many anti-igg of rabbit source P48 fusion.

The utility model has the following advantages:

(1) wrapping by the antigen P48 albumen of collaurum in the utility model is Mycoplasma bovis memebrane protein, has very strong specificity, can detect Mycoplasma bovis antibody truly, comprise serum antibody and whey antibody;

(2) the P48 albumen in the utility model is prokaryotic expression soluble protein, and purity is high, and active high, detect antibody sensitivity high, high specificity, has wide range of applications;

(3) cost is low, fast simple, convenient, without the need to specific installation and instrument, whole process completes in 15min, test cost-saving than indirect hemagglutination test and ELISA, test result presents macroscopic color reaction, without the need to specific apparatus, without the need to specific operating personnel, can conveniently for Mycoplasma bovis epidemiology survey and promote the use of in basic unit;

(4) label is stablized, and mark sample stores more than 2 year year at 4 DEG C, no signal relaxation phenomenon;

(5) collaurum is originally as redness, does not need to add chromogenic agents, and to human non-toxic's evil.

Accompanying drawing explanation

Fig. 1 is test strips schematic diagram of the present utility model, in figure: 1 absorption of sample pad, 2 colloidal gold pad, 3 detection line T, 4 nature controlling line C, 5 adsorptive pads, 6 base plates, 7 nitrocellulose membranes.

Fig. 2 be test strips of the present utility model to standard positive serum and negative serum testing result figure, wherein: 1 is the blank of sample diluting liquid; 2 is negative serum; 3 is Mycoplasma bovis positive standard serum; (negative serum and positive serum are first all diluted 10 times before detection, then detect by note: due to serum thickness).

Fig. 3 is the testing result figure of the utility model test strips for the positive standard serum of different gradient dilution, and 1 is the blank of sample diluting liquid; 2 is negative serum; 3 is that Mycoplasma bovis positive standard serum 1:10 dilutes; 4. be that Mycoplasma bovis positive standard serum 1:100 dilutes; 5 is that Mycoplasma bovis positive standard serum 1:1000 dilutes; 6 is that Mycoplasma bovis positive standard serum 1:10000 dilutes; 7 is that Mycoplasma bovis positive standard serum 1:100000 dilutes; 8 is that Mycoplasma bovis positive standard serum 1:200000 dilutes.

Fig. 4 is that the utility model test strips specific test detects, and is 1. Mycoplasma bovis positive standard serum; 2. Mycoplasma mycoides subsp.capri positive standard serum; 3. mycoplasma hyopneumoniae positive standard serum; 4. ox Pasteurella positive standard serum; 5. pleuropneumonia positive standard serum; 6. mycoplasma bovigenitalium positive standard serum; 7. mycoplasma ovine pneumoniae positive standard serum; 8. mycoplasma capri goat pneumonia subspecies positive standard serum; (serum is 1:10 dilution).

Embodiment

The utility model is below in conjunction with embodiment explanation.

1. the Expression and purification of P48 albumen

1.1 sequent synthesis and construction of recombinant plasmid

Mycoplasma bovis p48 gene (Genebank:NC_014760.1), the utility model is optimized this stream cipher, be optimized in colibacillary hobby according to codon, in this sequence of Mycoplasma bovis 4 are expressed the TGA(UGA of tryptophane simultaneously) be optimized to TGG(UGG), because this codon is terminator codon in Escherichia coli, add restriction enzyme site BamH I and Xho I at its two ends simultaneously;

Transfer to Invitrogen company to be synthesized in pet32a expression vector plasmid the p48 gene order after optimizing, after synthesis, carry out order-checking qualification, enzyme cuts qualification, will identify positive recombinant plasmid called after Pet32-a (+)-p48 successfully.

1.2P48 the expression of albumen

Recombinant plasmid pet32a (+)-p48 with genes of interest is transformed into competent cell BL21(DE3), single bacterium colony that picking 2 has been transformed into BL21 (DE3) is inoculated in 5ml respectively containing in the LB fluid nutrient medium of ampicillin, be positioned in 37 DEG C of shaking tables, about 220rpm concussion is cultured to OD value to 0.6, then the bacterium liquid drawing this 10ml is inoculated into the LB fluid nutrient medium of new 1L containing ampicillin 100ug/ml, at 16 DEG C, abduction delivering 20h under 0.01mmol/lIPTG concentration, collect thalline, resuspended with the PH7.2PBS of 100ml, and carry out ultrasonication 30min on ice.Then the liquid of ultrasonic process is placed in the centrifugal 30min of hydro-extractor 11000rpm, discards precipitation, collect supernatant 100ml.

The dialysis of 1.3 affinity column purifying P48 albumen and purifying protein

P48 recombinant protein is purified by nickel ion affinity chromatograph method.First by 5mL Ni-NTA His Bind Resin(purchased from Nanjing Jin Sirui biotechnology company) load empty chromatographic column, after allowing liquid lean on Action of Gravity Field naturally to ooze, the chromatographic column balance Buffer of 20 times of column volumes washs, then Sample supernatants is added in chromatographic column, then allows it flow out in conjunction with 60min on ice; The chromatographic column NTA-10 of 20 times of column volumes washs foreign protein; Then carry out wash-out with the NTA-60 wash-out Buffer of 6 column volumes respectively, every 5ml collects once, obtains albumen after purifying.

By the albumen of first volume purifying, the albumen of second volume purifying, three or four volume purifying protein merges, five or six volume purifying protein merges, and is respectively charged into bag filter, puts into the large beaker that 5L is equipped with dislysate, dislysate is 0.1MPB damping fluid, put into 4 DEG C of refrigerator dialysed overnight, within second day, take out, the P48 albumen of having dialysed is loaded EP pipe and puts into-40 DEG C of Refrigerator stores (1mL/ pipe).

2. the preparation process of colloidal gold strip

2.1 collaurum preparation process

Getting 1% aqueous solution of chloraurate 1ml adds in 100ml deionized water, boiling is heated at temperature control stirring device mouth, disposablely rapidly again add 1% trisodium citrate 1.5ml, continuous heating boils 15min, then namely obtains the colloidal gold solution of homogeneous transparent to 100ml with water polishing.

The preparation of 2.2 collaurum mark pads

The P48 recombinant protein bag of dialysing after the purifying obtained with said method is by collaurum and prepare colloidal gold pad, the pH value of antigen association colloid gold is 7.4, collaurum bag is 6ug/ml by the optium concentration of P48 albumen, the time of antigen coated gold grain is 30min, after antigen combines, add 20% BSA 50ul/ml close, off-period is 30 minutes, utilizes re-suspension liquid that colloid gold particle is resuspended after centrifugal, prepare collaurum mark pad, 37 DEG C of dryings 3 hours.Its re-suspension liquid formula is: 0.02M sodium tetraborate, 0.5% BSA, 3% sucrose, 0.5% casein, 0.5% polysorbas20.

The bag quilt of nature controlling line and detection line on 2.3 nitrocellulose filters

The utility model test strips nitrocellulose filter model is M135, buy in MILLIPORE (U.S.), bag is 100ug/ml affinity purification goat anti-ox two anti-igg by the antigen used of nitrocellulose filter detection line, wrap tested survey line 1ul/cm, the anti-ox of this affinity purification goat two is anti-to be bought in ImmunoReagents Reagent Company.

Bag is the many anti-igg of P48 albumen by nitrocellulose filter nature controlling line antigen, utilizes its concentration for 200ug/ml, wraps by nature controlling line 1ul/cm.This IgG obtains through purifying with hyper-immune serum.The preparation method of hyper-immune serum is as follows:

The preparation of Freund's complete adjuvant antigen used and incomplete Freund's adjuvant antigen in hyper-immune serum preparation, wherein: Freund's complete adjuvant antigen: Freund's complete adjuvant 3mL adds 1mg/mlP48 proteantigen 3mL; Incomplete Freund's adjuvant antigen: incomplete Freund's adjuvant 3mL adds 1mg/mlP48 albumen 3mL.

Above-mentioned adjuvant antigen is all prepared before immunity inoculation, and abundant mixing and emulsifying.

P48 albumen hyper-immune serum preparation process:

A. first, the healthy Male New Zealand White Rabbit at about 2kg, 12 monthly ages is selected.

B. get above-mentioned adequately emulsified Freund's complete adjuvant antigen, in every rabbit two back leg foot subcutaneous ， lymphonodi poplitei place and the inoculation of dorsal sc multiple spot, inoculation total amount is 1.5mL; Still inoculated by dorsal sc multiple spot with Freund's complete adjuvant antigen 1 .5ml after 14 days, carry out second time immunity; After 14 days, with above-mentioned incomplete Freund's adjuvant antigen, by every rabbit sole two point, dorsal sc multiple spot is inoculated, and inoculation total amount is 3mL, is its third time immunity;

C. after 14 days by ear edge vein exploitating blood separation of serum, with IHA test examination blood, immunize rabbit serum I HA tires and reaches 1:512, formally takes a blood sample, and the centrifugal 5min separation of serum of 3000rpm, is P48 albumen hyper-immune serum.

D. by saturated ammonium sulphate method the serum be separated to purified and obtain the many anti-igg of P48 albumen.

2.4 test strips assemblings

Operation is carried out will comprising the nitrocellulose filter of detection line and nature controlling line respectively from top to bottom as follows on PVC backer board, colloidal gold pad, sample pad, and absorption pad assembles in order, PVC liner plate is located at bottommost, stage casing, liner plate top is provided with nitrocellulose filter, nitrocellulose filter upper left end posts absorption pad, nitrocellulose filter upper right end is provided with gold mark pad, the upper right end of gold mark pad is provided with sample pad, with spray gun, the many anti-igg of P48 of aforementioned affinity purification goat anti-ox two anti-igg that obtains and purifying are made detection line and nature controlling line on nitrocellulose filter respectively again, in the utility model test strips, detection line and nature controlling line line-to-line distance remain on 0.8mm.Test paper is cut into the wide strip of 4mm again, namely make the colloidal gold strip schematic diagram of the structure detection Mycoplasma bovis of the present utility model antibody as accompanying drawing 1.

Test strips principle of the present utility model is that dual-antigen sandwich method detects antibody, and wherein criterion is under the prerequisite of nature controlling line colour developing, and detection line colour developing is positive; Detection line, without colour developing, is negative.If nature controlling line does not develop the color, then lost efficacy, and needed to redeterminate.

The utility model colloidal gold strip detects positive serum and negative serum respectively, the results are shown in accompanying drawing 2, and from accompanying drawing 2, positive serum display detection line and nature controlling line two lines are redness; Negative nature controlling line colour developing, detection line does not develop the color; It is nature controlling line colour developing that blank is only sample diluting liquid result, and detection line does not develop the color.Wherein sample diluting liquid is the 0.1MPBS damping fluid containing 0.5% polysorbas20.

The utility model colloidal gold strip detects the different dilutability of positive serum, the results are shown in accompanying drawing 4, and from accompanying drawing 3, the equal nature controlling line colour developing of negative serum, sample diluting liquid blank, detection line does not develop the color.Positive serum 1:10,1:100,1:1000,1:10000,1:100000 are nature controlling line and detection line all develops the color, and presents the positive; And positive serum 1:200000 only nature controlling line colour developing, present feminine gender, therefore in this invention, test strips sensitivity can reach, and the Mycoplasma bovis indirect hemagglutination detection method set up before only can detect 1:4096, so this invention substantially increases detection sensitivity.

The utility model test strips specific test testing result is shown in accompanying drawing 4, wherein have detected Mycoplasma bovis positive standard serum, Mycoplasma mycoides subsp.capri positive standard serum, mycoplasma hyopneumoniae positive standard serum, ox Pasteurella positive standard serum, pleuropneumonia positive standard serum, mycoplasma bovigenitalium positive standard serum, mycoplasma ovine pneumoniae positive standard serum and mycoplasma capri goat pneumonia subspecies positive standard serum, result only presents the positive by Mycoplasma bovis positive serum, all the other similar mycoplasmas and the pathogenic autoantibody of ox similar conditions can be caused all to present feminine gender, prove that this test strips specificity is good, reliable results.

Import ELISA kit and detection paper of the present utility model is utilized to contrast, detect the negative sample (containing whey, serum) totally 83 parts of clear and definite background, positive (containing whey, serum) totally 189 parts, wherein import ELISA kit detects negative serum 90 parts, positive serum 182 parts, and test strips testing result negative serum 86 parts in this invention, positive serum 186 parts.Detect with import ELISA kit and contrast, negative match-rate is 95.6%, positive coincidence rate is 97.8%, and import ELISA kit detects the cost of sample up to 5 yuan/part, and action need 3 hours just can go out testing result, and test strips cost 0.5 yuan/part in the utility model, in 15min, only just can present result that is clear, that understand, greatly reduce cost, improve efficiency.

Claims (1)

1. one kind is detected the test strips of Mycoplasma bovis antibody, this test strips by base plate, be arranged on base plate that linearly aligned absorption of sample pad, the colloidal gold pad being coated with antigen, nitrocellulose membrane and adsorptive pads form successively, nitrocellulose membrane is coated with nature controlling line and detection line, it is characterized in that: the antigen of colloidal gold pad bag quilt is Mycoplasma bovis P48 fusion, detection line bag quilt be goat anti-ox two anti-igg, nature controlling line is many anti-igg of rabbit source P48 fusion.