CN102128926A - Double-adaptor kit and use thereof of in detection of tubercule bacillus antigen - Google Patents
Double-adaptor kit and use thereof of in detection of tubercule bacillus antigen Download PDFInfo
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- CN102128926A CN102128926A CN2010105744255A CN201010574425A CN102128926A CN 102128926 A CN102128926 A CN 102128926A CN 2010105744255 A CN2010105744255 A CN 2010105744255A CN 201010574425 A CN201010574425 A CN 201010574425A CN 102128926 A CN102128926 A CN 102128926A
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Abstract
The invention discloses a novel double-adaptor sandwich enzyme-linked immuno sorbent assay (ELISA) kit for sensitive and quick diagnosis of tubercule bacillus thallus antigen, which comprises two adaptors, namely ZXL2 and ZXL3. In the invention, the DNA single-chain adaptors screened by using systematic evolution of ligands by exponential enrichment (SELEX) are named ZXL2 and ZXL3, the double-adaptor sandwich ELISA kit is built for the first time, the kit is combined with the early diagnosis of tuberculosis, and compared with the conventional acid-fast stain detection method used in clinic, the specificity is improved by 35 percent and the sensitivity is improved to 100CFU/ml from the original 10,000CFU/ml. The diagnosis time is reduced to 1 day from the original 2 to 8 days required by the conventional diagnosis of sputum culture. In the invention, the operation is simple and quick, the cost is low, the specificity, sensitivity and accuracy of the diagnosis of patients with tuberculosis are improved greatly, and particularly the novel tubercule bacillus antigen early diagnosis kit, which has a bright clinic application prospect, is provided.
Description
Technical field
The invention belongs to the molecular immunology field.Be specifically related to a kind of can specificity in conjunction with virulent mycobacteria H37Rv somatic surface antigen, and not in conjunction with single-strand DNA aptamer kit and the application in tuberculosis antigen detects thereof of BCG.
Background technology
Tuberculosis is brought serious threat by the chronic expendable infectious disease that Much's bacillus causes, the people beast suffers from altogether to human health.Show that according to The World Health Organization's statistical data the whole world has nearly 1/3rd people to infect tubercle bacillus at present, the active tuberculosis patient reaches 2,000 ten thousand, has every year 3000000 people to die from tuberculosis, annual neopathy number 800～10,000,000.1993, the World Health Organization (WHO) announced " global tuberculosis is in a state of emergency ", tuberculosis was classified as one of infectious disease of emphasis control.
China is the high burden of tuberculosis country, existing active tuberculosis patient 5,000,000 people, infectiousness pulmonary tuberculosis patient 2,000,000 people, annual newly-increased about 1,300,000 people of case, because of (" national tuberculosis prevention and treatment planning (2001-2010) ", the Office of the State Council sends out file [2001] No. 75) more than dead 150,000 people of tuberculosis.State Council has determined that tuberculosis is one of China's three big keypoint control infectious diseases.The order of severity of China's tuberculosis epidemic situation is only second to India, occupies the second in the world.It is three high one low that China's tuberculosis epidemic situation is, i.e. morbidity rate height, mortality ratio height, resistant rate height, and year degradation rate is low.Because popular, the movement of population of HIV/AIDS, resistance tulase increase year by year, and incidence of tuberculosis begins to rise, the tuberculosis epidemic situation is more serious in recent years.
Use novel diagnostic method that tuberculosis is carried out early diagnosis and treatment has crucial meaning, there are many defectives in the existing diagnostic techniques of tuberculosis both at home and abroad at present, lack special, effective, quick, convenient, cheap diagnostic techniques and product, particularly lack responsive early detection means always tuberculosis antigen.Past PPD(purified protein derivative of tuberculin) also be usually used in diagnosis lungy and epidemiology survey, but it lacks specificity, susceptibility is low, and certain application limitation is arranged; The sensitivity of traditional phlegm acid-fast stain is difficult to reach, and can not distinguish different Much's bacillus; There is the window phase problem in traditional antibody detection method, can not reach testing goal, and can not distinguish the BCG vaccination or the problem of infection tuberculosis; Though the diagnostic method high specificity of Fa Zhan RT-PCR needed expensive instrument, the cost height in recent years.Clinically to the patient of doubtful infection tuberculosis, owing to lack effective diagnostic method, usually adopt first antituberculosis therapy, the effect with treatment serves as according to diagnosing again, wasted lot of manpower and material resources thus, also may cause mistaken diagnosis or delay treatment other diseases.So, explore novel diagnostic method to tuberculosis carry out early diagnosis research be used for tuberculosis fast, effectively, convenient, cheap specific detection, the acid-fast stain method used of tradition is sensitiveer and be used for the novel agent of tubercle bacillus antigen diagnosis apace clinically particularly to be badly in need of a kind of ratio of exploitation, for timely diagnosis, treatment tuberculosis patient with control the tuberculosis epidemic and all have crucial meaning.
Summary of the invention
At above-mentioned deficiency, the invention provides a kind of can specificity in conjunction with two kinds of single-strand DNA aptamer kits of toxicity Much's bacillus H37Rv.
The present invention obtains two adaptive sons of dna single chain by the SELEX triage techniques by multi-turns screen, difference called after ZXL2 and ZXL3, and its nucleotide sequence is respectively shown in SEQ ID No.1 and SEQ ID No.2.ZXL2 and ZXL3 have good specificity to Much's bacillus H37Rv.
Based on this, the invention provides a kind of two adaptive sub-detection kit, it comprises adaptive sub-ZXL2 and ZXL3.
Mentioned reagent box, a kind of bag among adaptive sub-ZXL2 and the ZXL3 be by in ELISA Plate, the another kind biotin labeling.Preferred ZXL2 wraps quilt in ELISA Plate, the ZXL3 biotin labeling.
The mentioned reagent box, it also can further comprise in enzyme mark Streptavidin, PBST and the stop buffer one or more.Described enzyme mark Streptavidin is the horseradish peroxidase-labeled Streptavidin.Described stop buffer is the concentrated sulphuric acid, and preferred stop buffer is the H of 2M
2SO
4
The present invention combines adaptive son of this strand and diagnosis lungy first, and this adaptive sub-ZXL2, ZXL3 are external synthetic easily, and can specificity and Much's bacillus H37Rv tuberculosis.Kit of the present invention than the advantage of the diagnostic method of traditional phlegm acid-fast stain is: kit of the present invention can distinguish the toxicity Much's bacillus and other mycobacterium comprises BCG, and can detect and be lower than 10
4The tulase of CFU/ml, and the sensitivity of traditional phlegm acid-fast stain is difficult to reach; The advantage of comparing with traditional antibody detection method is: can detect antigen, and can shorten window phase, reach the early diagnosis purpose; The advantage of comparing with the diagnostic method of RT-PCR is: do not need expensive instrument, cost is low.And the adaptive daughter of dna single chain is synthetic very simple outward, and cheap, our method of invention is simple to operate simultaneously, the running time short (finishing in 1 day), the diagnostic sensitivity and the degree of accuracy of cultivating the positive for tuberculosis patient phlegm are all very high, and potential applicability in clinical practice is wide.
Description of drawings
Determining of the two adaptive sub-concentration of adaptive sub-sandwich method of Fig. 1
Two kinds of adaptive sons combine with H37Rv, BCG respectively, and the adaptive sub-ZXL2 of the abiotic mark of 100nM is in conjunction with bacterium, when the biotin labeled adaptive sub-ZXL3 of 10nM detects, and the minimum (OD of nonspecific reaction
450＜0.2), the optium concentration of definite adaptive son of ZXL2 that combines with tubercle bacillus H37Rv is 100nM, and the optium concentration of the adaptive son of ZXL3 is 10nM.
The specificity of the two adaptive sub-kits of Fig. 2 the present invention is identified
Among the figure 1, H37Rv; 2, BCG; 3, mycobacterium smegmatis; 4, mycobacterium avium; 5, Mycobacterium intracellulare; 6, micrococcus luteus; 7, Pseudomonas aeruginosa; 8, golden yellow grape ball; 9, Escherichia coli; 10, staphylococcus aureus persister; 11, white staphylococcus.
Two adaptive sub-sandwich methods can be PBS at environment, and bacterial concentration is 10
4Effectively identify H37Rv and different non-branch bacillus and mycobacterium under the condition of CFU/mL.
The two adaptive sub-sandwich methods of Fig. 3 detect normal person and patient's sputum
When normal person's sputum uses two adaptive submethods to detect, OD
450Value＜0.1.The tuberculosis patient sputum acid-fast stain positive, can both react with the adaptive son of the ZXL3 of adaptive son of the ZXL2 of 100nM concentration and 10nM concentration, and very high OD value is arranged.OD
450Value＞0.2.
Embodiment
Following examples are used to further specify the present invention, but should not be construed as limitation of the present invention.If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.Below every operation that relates to tubercle bacillus, all in Biohazard Safety Equipment, operate.Abiotic plain mark promptly refers to unmarked among the present invention.
Determining of the adaptive sub-concentration of embodiment 1 kit
Experimental technique
(1) the adaptive son of biotin labeled ZXL3 that synthesizes from company is diluted to 10nM respectively with aseptic distilled water, 40nM, 100nM, the concentration of 400nM is preserved in-20 ℃ of refrigerators.
(2) bacterium not of the same race: tubercle bacillus standard strain H37Rv, Bacille Calmette-Guerin, yellow micrococcus luteus, Pseudomonas aeruginosa, staphylococcus aureus, bacillus enteritidis, staphylococcus aureus persister, staphylococcus albus is cultivated in proper culture medium respectively, tubercle bacillus standard strain H37Rv wherein, Bacille Calmette-Guerin (BCG) is cultivated in modified Russell medium, and other bacteriums are cultivated in the LB nutrient culture media, using the same day then, with the different concentration of physiological saline furnishing.
(3) evaluation of two adaptive sub-method optimum concentrations, with 100nM, the unlabelled adaptive sub-ZXL2(0.05mol/L NaHCO of 400nM
3, the pH value is 8.0 dilutions) bag is by behind 96 orifice plates, under ultraviolet light, expose normal temperature and spend the night.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), to wash 6 times, every hole adds variable concentrations (1 * 10
5CFU/ml, 1 * 10
4CFU/ml, 1 * 10
3CFU/ml, 1 * 10
2CFU/ml, 1 * 10
1CFU/ml) H37RV, BCG 100 μ l, each concentration is established 6 multiple holes, establishes a negative control hole that does not add bacterium simultaneously, and 4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST, wash 6 times, because each concentration of every kind of bacterium is established 8 multiple holes, so the adaptive son of 2 multiple Kong Jiayi concentration of each concentration of two kinds of bacteriums, every hole 100 μ l, 4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, add the biotin labeled adaptive sub-ZXL3(10nM of variable concentrations, 40nM, 100nM, 400nM), and 4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, add the Streptavidin that adds horseradish peroxidase-labeled then, 37 ℃, hatch half an hour.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, use the H of 2M then
2SO
4Cessation reaction.On microplate reader, read the OD value with the optical filter of 450nm.
Experimental result: the result as shown in Figure 1, two kinds of adaptive sons combine with H37Rv, BCG respectively, the unlabelled adaptive sub-ZXL2 of 100nM is in conjunction with bacterium, when the biotin labeled adaptive sub-ZXL3 of 10nM detects, the minimum (OD of nonspecific reaction
450＜0.2), the optium concentration of definite adaptive son of ZXL2 that combines with tubercle bacillus H37Rv is 100nM, and the optium concentration of the adaptive son of ZXL3 is 10nM.
The specific evaluation of 2 pairs of adaptive sub-methods of embodiment
It is 10 that H37Rv, BCG, mycobacterium smegmatis, mycobacterium avium, Mycobacterium intracellulare, micrococcus luteus, Pseudomonas aeruginosa, staphylococcus aureus, staphylococcus aureus persister, Escherichia coli, white staphylococcus are diluted to concentration
4CFU/mL, standby.
Adaptive sub-ZXL2(0.05mol/L NaHCO with the abiotic plain mark of 100nM
3, the pH value is 8.0 dilutions) bag is by behind 96 orifice plates, under ultraviolet light, expose normal temperature and spend the night.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), to wash 6 times, every hole adds 1 * 10 respectively
3The bacterium that CFU/ml(is different, same concentration) different bacterium 100 μ l, every kind of bacterium is established 2 multiple holes, establishes a negative control hole that does not add bacterium simultaneously, and 4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST, washes 6 times, adds 10nM and goes into biotin labeled adaptive sub-ZXL3,4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, add the Streptavidin that adds horseradish peroxidase-labeled then, 37 ℃, hatch half an hour.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, use the H of 2M then
2SO
4Cessation reaction.On microplate reader, read the OD value with the optical filter of 450nm.
The result as shown in Figure 2, two adaptive sub-sandwich methods can be PBS at environment, bacterial concentration is 10
4Effectively identify H37Rv and different non-branch bacillus and mycobacterium under the condition of CFU/mL.
The comparison of 3 pairs of adaptive sub-method clinical diagnosises of embodiment and Ziehi-Neelsen stain
Materials and methods
(1). get 97 portions of normal person's sputums, use 3%NaHCO
3Proportional diluted.Normal person's sputum is collected from five universities of Wuhan Area: preclinical medicine institute of Wuhan University (20 parts), our department of Wuhan University (20 parts), Central China Normal University's (20 parts), Wuhan University of Technology's (17 parts), Hua Zhong Agriculture University's (20 parts), take voluntary form, randomly draw the age at 97 portions of non-tuberculosis infection phase normal person's sputums of 18 to 27 years old.Boy student 74 people wherein, schoolgirl 23 people; 59 people are from the rural area, and 38 people are from the city; 42 people confirm to inoculate Bacille Calmette-Guerin, and all the other 55 people do not inoculate or the unclear Bacille Calmette-Guerin of whether inoculating; In the 6 people families tubercular is arranged; 4 people are once by the tuberculosis infection mistake.
(2). get the sputum of 48 parts of tuberculosis patients, a part is taken away and is done acid-fast stain, another part 3%NaHCO
3Proportional diluted is carried out following two adaptive sub-methods and is detected.
(3). with the unlabelled adaptive sub-ZXL2(0.05mol/L NaHCO of 100nM
3, pH value is 8.0 dilutions) bag is by behind 96 orifice plates, under ultraviolet light, expose normal temperature and spend the night.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), to wash 6 times, every hole adds normal person and the patient's sputum 100 μ l after the dilution respectively, establishes one simultaneously not with the negative control hole of sputum, and 4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST, washes 6 times, adds 10nM and goes into biotin labeled adaptive sub-ZXL3,4 ℃, overnight incubation.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, add the Streptavidin that adds horseradish peroxidase-labeled then, 37 ℃, hatch half an hour.Discard the liquid in the hole, every hole adds 300 μ l PBST(0.5 ‰), wash 6 times, use the H of 2M then
2SO
4Cessation reaction.On microplate reader, read the OD value with the optical filter of 450nm.
Experimental result
Table 1
| ? | Total coincidence rate (%) | Specificity (%) |
| Two adaptive sub-sandwich methods | 88.28 | 87.37 |
| Acid-fast stain | 51.97 | 51.87 |
The tuberculosis patient that disease rescue centre in Wuhan City's is made a definite diagnosis compares as the positive, and the normal person as feminine gender relatively calculates total coincidence rate, the specificity of two adaptive sub-sandwich methods and acid-fast stain.The result shows in the table: total coincidence rate of two adaptive sub-sandwich methods is 88.28%, specificity is 87.37%.With the degree of conformity of acid-fast stain be 0.988; Total coincidence rate of acid-fast stain is 51.97%, specificity is 51.87%.According to the specificity height of this presentation of results this method than acid-fast stain.Two adaptive sub-sandwich methods and acid-fast stain detection method compare, and specificity improves 35 percentage points, and susceptibility is brought up to 100 CFU/mL by 10000 original CFU/mL.With diagnostic method 2-8 week being reduced to 1 day from past of tradition by the phlegm cultivation.
As shown in Figure 3, when normal person's sputum uses two adaptive submethods to detect, OD
450Value＜0.1.The tuberculosis patient sputum acid-fast stain positive, can both react with the adaptive son of the ZXL3 of adaptive son of the ZXL2 of 100nM concentration and 10nM concentration, and very high OD value is arranged.OD
450Value＞0.2.
Embodiment 4 kits of the present invention are formed
1. bag is by the ELISA Plate of ZXL2;
With the phosphate buffer of pH8.0 the adaptive sub-ZXL2 of abiotic plain mark being diluted to concentration is 100 nM.By 96 hole elisa plates, 4 ℃ of bags are spent the night according to every hole 100ml bag.
2. biotin labeling ZXL3(is synthetic by match Parkson, Shanghai company)
3. horseradish peroxidase-labeled Streptavidin;
4. substrate colour developing liquid:
A liquid: TMB 0.02%(w/v)
B liquid: anhydrous citric acid 2.1g, anhydrous Na
2HPO
42.82g, 0.75% hydrogen peroxidase 10 .64ml, distilled water is settled to 100ml
Collocation method: take by weighing TMB 20mg (solid), earlier with absolute ethyl alcohol 10ml dissolving, fully vibration (it is clean that reagent bottle is wanted, drying) adds the distilled water constant volume to 100 ml, and this is a substrate A. substrate B, get anhydrous citric acid 2.1g, anhydrous Na
2HPO
42.82g 0.75% hydrogen peroxidase 10 .64ml, distilled water are settled to 100ml (need not adjust pH, should in the 4.5-5.0 scope).A:B respectively gets the 5ml mixing during use.(it is Bioisystech Co., Ltd that composite is ordered in reaching section)
5. stop buffer: 2M H
2SO
4
Annotate:
The preparation method of used LB nutrient culture media is among the present invention: the consumption of preparation 1L: peptone 10g yeast extract 5g NaCl 10g adjust pH to the 7.0-7.2. packing in 121 degrees centigrade of sterilization 15min.
The preparation of used modified Russell medium is sent out method and is among the present invention: monosodium glutamate (sodium glutamate is more than 95%) 7.2g; Potassium dihydrogen phosphate 2.4g; Magnesium sulphate 0.24g; Magnesium citrate 0.6g; Glycerine 12mL; Distilled water 600mL; Farina 30g; Whole egg liquid 1000mL; 2% peacock green 20mL. preparation method: after the dissolving of each salt constituents, add farina, mixing boils 30～40min (shaking anti-grumeleuse therebetween frequently) in the boiling water pot, in the pasty state, treat cold after, add the fresh whole egg liquid 1000mL that filters through antiseptic gauze, mixing.Add 2% peacock green 20mL, mixing, the packing test tube (18mm * 180mm).Each test tube adds nutrient culture media 7mL, and the medium slant height is advisable for 2/3rds places of cultivating fiduciary point test tube bottom, puts in the serum coagulator and solidifies.During temperature to 90 ℃, put into the packing test tube, in the coagulator to put two-layer being advisable.Treat 85～90 ℃ of coagulator Nei Wenduda, timing is taken out after solidifying 1～1.5h, puts coldly, and it is standby to put 4 ℃ of refrigerators after the sterility test, uses in one month.
Used bag is cushioned liquid (pH8.0,0.05M carbonate buffer solution): Na among the present invention
2CO
31.59 gram, NaHCO
32.93 the gram adding distil water to 1000ml, disposes before using.
Sequence table
＜110〉Wuhan University
＜120〉a kind of preparation of two adaptive sub-sandwich ELISA kits and the application in the tubercle bacillus antigen diagnosis thereof
<130>
<160> 2
<170> PatentIn?version?3.5
<210> 1
<211> 30
<212> DNA
＜213〉artificial sequence
<400> ZXL2
TTTAATCACA?CAACACCGTG?TTTCAAGCTT 30
<210> 2
<211> 30
<212> DNA
＜213〉artificial sequence
<400> ZXL3
CGACAGGGGT?CTGAATCACG?TACATTCGTA 30
Claims (7)
1. two sandwich kit of adaptive son comprises adaptive sub-ZXL2 of DNA and ZXL3, and their nucleotide sequence is respectively shown in SEQ ID No.1 and SEQ ID No.2.
2. kit according to claim 1 is characterized in that, a kind of bag among ZXL2 and the ZXL3 is by in ELISA Plate, the another kind biotin labeling.
3. kit according to claim 2 is characterized in that, ZXL2 wraps quilt in ELISA Plate, the ZXL3 biotin labeling.
4. according to each described kit of claim 1 ~ 3, it also comprises in enzyme mark Streptavidin, PBST and the stop buffer one or more.
5. kit according to claim 4 is characterized in that, described enzyme mark Streptavidin is the horseradish peroxidase-labeled Streptavidin.
6. kit according to claim 4 is characterized in that, described stop buffer is the H of 2M
2SO
4
7. the application of each described kit of claim 1 ~ 6 in detecting tubercle bacillus antigen.
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|---|---|---|---|
| CN2010105744255A CN102128926A (en) | 2010-12-06 | 2010-12-06 | Double-adaptor kit and use thereof of in detection of tubercule bacillus antigen |
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| Publication Number | Publication Date |
|---|---|
| CN102128926A true CN102128926A (en) | 2011-07-20 |
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|---|---|---|---|
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012483A (en) * | 2007-02-02 | 2007-08-08 | 浙江大学 | Checking reagent containing nucleic acid gamete and method for making same and use |
| CN101109013A (en) * | 2006-07-20 | 2008-01-23 | 武汉大学 | Micromolecule nucleotide DNA adaptorprotein against tubercle bacillus infection and method of preparing the same |
-
2010
- 2010-12-06 CN CN2010105744255A patent/CN102128926A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109013A (en) * | 2006-07-20 | 2008-01-23 | 武汉大学 | Micromolecule nucleotide DNA adaptorprotein against tubercle bacillus infection and method of preparing the same |
| CN101012483A (en) * | 2007-02-02 | 2007-08-08 | 浙江大学 | Checking reagent containing nucleic acid gamete and method for making same and use |
Non-Patent Citations (2)
| Title |
|---|
| JEEVALATHA,V: "Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay", 《LABORATORY INVESTIGATION》 * |
| 甄蓓: "体外筛选炭疽芽孢适配子", 《生物化学与生物物理学报》 * |
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Application publication date: 20110720 |