CN101650365A - Method and kit for quickly detecting mycobacterium tuberculosis - Google Patents

Method and kit for quickly detecting mycobacterium tuberculosis Download PDF

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Publication number
CN101650365A
CN101650365A CN200810043709A CN200810043709A CN101650365A CN 101650365 A CN101650365 A CN 101650365A CN 200810043709 A CN200810043709 A CN 200810043709A CN 200810043709 A CN200810043709 A CN 200810043709A CN 101650365 A CN101650365 A CN 101650365A
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bacteriophage
tubercle bacillus
weight
sample
liquid
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CN200810043709A
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CN101650365B (en
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胡志能
王亚新
吴伟清
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Alere Shanghai Diagnostics Co Ltd
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Inverness Medical Shanghai Co Ltd
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Abstract

The invention discloses a method for quickly detecting mycobacterium tuberculosis, which comprises the following steps: 1, infecting phage with the mycobacterium tuberculosis; 2, killing the phage notinfecting outside the mycobacterium tuberculosis; 3, amplifying the infecting phage; and 4, after labeling gold with antiphage antibody, adopting quick immunochromatography to detect whether the phage exists or not so as to judge whether the mycobacterium tuberculosis exists in a sample. In addition, the invention also discloses a corresponding kit, which comprises sample treating fluid, proliferous liquid, a killing agent, mycobacterium smegmatis, mycobacteriophages and a phage colloidal gold method immunity-chromatography test pen. The method and the kit can quickly and accurately detect the mycobacterium tuberculosis so as to meet the requirement of quickly diagnosing pulmonary tuberculosis and timely medicate the patient.

Description

Method of a kind of fast detecting tubercle bacillus and kit
Technical field
The present invention relates to molecular biology, immunology and protein detection technology field.More specifically, the method and the kit that relate to a kind of fast detecting tubercle bacillus.
Background technology
Pulmonary tuberculosis (Tuberculosis) is the chronic infectious disease of a kind of infecting both domestic animals and human of being caused by tubercle bacillus (Tubercle Bacillus), is present single infective bacterial causing death's principal element.In recent years, along with increasing and the appearance of multiple drug resistance bacterial strain of popular, the multiple malignant tumor patient of acquired immune deficiency syndrome (AIDS), increased the difficulty in the control of tubercle bacillus affection disease especially.Statistics shows, there is tuberculosis germ the infected 2,000,000,000 (accounting for total population 1/3) in the whole world, infects a people p.s., annual new 8,000,000 people that infect of global tuberculosis germ, if do not take the non-proliferation measure immediately, tuberculosis will be seized more than 3,000 ten thousand people's life in 10 years from now on.China whole nation tuberculosis infection person 3.3 hundred million, tuberculosis patient 6,000,000, annual patient because of tuberculosis death is the twice of the dead summations of all kinds of infectious diseases up to 250,000, therefore China is upgraded to Category B notifiable disease with tuberculosis by the Class C infectious disease, lists the strict control category in.According to WHO statistics, the whole world has 5,000 ten thousand to 100,000,000 people to infect tuberculosis every year, and about three million peoples die from tuberculosis, wherein more than 80% in developing country.Therefore, how to diagnose and treat the key link that becomes the diffusion of control tuberculosis and propagate fast as soon as possible.
At present, phthisical diagnosis mainly is by getting sputum examination except general chest X-ray examination, comprises that acid-fast bacilli sputum smear dyeing microscopy and tubercle bacillus cultivate, and the bacteriology checking of tubercle bacillus is to make a definite diagnosis phthisical important evidence.Acid-fast bacilli plate coating checking susceptibility is low, poor specificity, and can't determine that bacterium is anyway.Tubercle bacillus is cultivated normally with the Russell medium of specimen inoculation to be checked to improvement, cultivates 20~60 days, and treats comprehensively to judge according to biological property and biochemical reaction many index behind the bacterial growth again for 37 ℃.Since the thalline complex structure of tulase, rich lipid, and hydrophobicity makes that pulmonary bacillus (Mycobacterium Tuberculosis) speed of growth is slow, just divides once every 12~twenty four hours.So, utilize cultural method to detect the existence of tulase, often need the time in three~eight weeks just can obtain the result, length expends time in, and complex operation, can't satisfy and make a definite diagnosis phthisical needs fast, often the opportunity of delay treatment and susceptibility not high (positive rate only about 40%).
Recent years, bacteriophage rapid amplifying method is utilized the quick growth characteristics of mycobacterium smegmatis (MycobacteriumSmegmatics) and the relative specificity of D29 bacteriophage, carries out the fast detecting of tubercle bacillus.The core content of this method is:
A. handled sample to be checked 30 minutes, the centrifugal supernatant that goes with sample preparation liquid chamber temperature;
B. increased bacterium 24 hours with 37 ℃ of enrichment liquids;
C. add 37 ℃ of incubations of D29 mycobacteriophage 1 hour, and infected the tubercle bacillus thalline;
D. do not invade the bacteriophage of tubercle bacillus with ending the liquid deactivation, room temperature effect 10 minutes is with aborting infection;
E. get the liquid to be measured of steps d gained, drip and to wrap in advance on the agar bacterium colony double dish of quilt in mycobacterium smegmatis, 37 ℃ incubation 15-20 hour;
F. according to the formation situation result of determination of plaque on the bacterium colony double dish.
The kit high specificity of this method, result are distinct, need not specific apparatus, can be used for the tubercle bacillus thalline Rapid identification of living in the various samples to be checked; But whole test needed just can finish at least in 48 hours, and operation is complicated, and sensitivity is also not high enough.
In recent years, increasingly mature along with Protocols in Molecular Biology, antigen purification technology and monoclonal antibody technique, some fast the method for inspection come out one after another, as poly synthase chain reaction method (PCR), ELISA method, immunity percolation method and immunochromatographic method.
Polymerase chain reaction,PCR (PCR) is the external enzymatic amplification technique that is mediated mycobacterium tuberculosis dna particular sequence fragment in the sample to be checked by a pair of specific Oligonucleolide primers, circulation by three different temperatures, dozens of sex change, renaturation, extension, the particular target sequence can be amplified millions of times, but and show its fast, characteristics such as high sensitivity quantitative test and theoretic high specific.But this method is operated more complicated, and higher to operating equipment and personnel's technical requirement, the laboratory easily produces pollution.
Enzyme linked immunosorbent assay (ELISA), immunity percolation method (DIGFA) and immunochromatographic method (ICT) all are based on the detection method that sero-immunity grows up.These methods all are that the characteristic antigen with tubercle bacillus is catches, with the anti-human immunoglobulin(HIg) of mark or SPA (a kind of surface protein (single chain polypeptide) of aureus cell wall) etc. is indication mechanism, detect the against mycobacterium tuberculosis circulating antibody that exists in patient's body, indirectly patient's ill situation is diagnosed.Enzyme linked immunosorbent assay needs the instrument of specialty and the professional of high operative skill.Immunity percolation method and immunochromatographic method detect required time and also shorten dramatically all without any need for instrument, and be easy and simple to handle, visual result.But the common problem of said method is sensitivity and specificity is difficult to reach requirement, can not distinguish the outer tuberculosis of pulmonary tuberculosis and lung, and can not distinguish the case that infection has once been cured; The product that has even can not distinguish the case of injecting Bacille Calmette-Guerin (BCG).
Summary of the invention
The technical problem to be solved in the present invention provides the method for a kind of fast detecting tubercle bacillus, it is fast inadequately that this method has overcome existing bacteriophage rapid amplifying method, the defective that serological diagnostic method medium sensitivity and specificity are not high, diagnostic procedure was tapered in 30 hours, and sensitivity and specificity have been improved, make a definite diagnosis phthisical needs fast to satisfy, so that to the timely medication treatment of patient.For this reason, the present invention also provides the kit of a kind of fast detecting tubercle bacillus.
In a first aspect of the present invention, the method for a kind of fast detecting tubercle bacillus is provided, comprise the steps: the first step, use the phage-infect tubercle bacillus; In second step, kill tubercle bacillus and do not infect bacteriophage outward; The 3rd step, the bacteriophage that amplification has been infected; In the 4th step, with phage-resistant antibody standard gold after, thereby adopt the existence of fast immune chromatographic method detection bacteriophage whether in the judgement sample whether tubercle bacillus to be arranged.
The 4th step specifically comprised:
(1). prepare special monoclonal antibody at bacteriophage;
(2). special monoclonal antibody is attached to the colloid gold particle of activation;
(3). preparation bacteriophage colloidal gold method immune chromatograph testing pen, p-wire and control line are set on test-strips, with the control detection quality, detect with bacteriophage colloidal gold method immune chromatograph testing pen, whether tubercle bacillus is arranged in the judgement sample.
The 3rd step adopted the liquid cultivating method amplification to infect the bacteriophage of tubercle bacillus, adopts Michaelis 7H9 liquid nutrient media and add the amplification host of shame dirt bacillus as bacteriophage in amplification procedure.
In a second aspect of the present invention, the kit of a kind of fast detecting tubercle bacillus is provided, this kit comprises sample preparation liquid, enrichment liquid, agent for killing, mycobacterium smegmatis, mycobacteriophage, bacteriophage colloidal gold method immune chromatograph testing pen.
Described sample preparation liquid is 2~4% sodium hydroxide solution; Described enrichment liquid is by Michaelis 7H9 nutrient culture media 5-7 weight portion, lime chloride 1-2 weight portion, ampicillin 25-50 weight portion, amphotericin B 25-50 weight portion, bovine serum albumin(BSA) 100-200 weight portion, glucose 10-15 weight portion, oleic acid 1-2 weight portion and hydrogen peroxidase 0.1-0.2 weight portion, all is dissolved in the pure water of 1000 weight portions being prepared from; Described agent for killing is 5~8% ferrous sulphate ammonia solution; Described mycobacterium smegmatis is the 3~5mg/ml mycobacterium smegmatis that is dissolved in Michaelis 7H9 fluid nutrient medium; Described mycobacteriophage is the mycobacterium D29 bacteriophage that is dissolved in Michaelis 7H9 fluid nutrient medium; After described bacteriophage colloidal gold method immune chromatograph testing pen was put into the plastic housing closure by the biological test chip, the aluminium foil bag of putting into a suitable size again was sealed to form.
As shown in Figure 1, this biological test chip comprises collaurum pad 4, p-wire (T line) 5, control line (C line) 7, sample pad 3, PVC base plate 13, NC film (nitrocellulose filter) 6, absorbent filter 8.Wherein be coated with the compound of anti-gp17 monoclonal antibody (a kind of coat protein of D29 bacteriophage) colloid gold above the collaurum pad 4, be coated with p-wire (T line) 5 and control line (C line) 7 above the NC film (nitrocellulose filter) 6, be coated with anti-gp23 monoclonal antibody (the another kind of coat protein of D29 bacteriophage) on the p-wire (T line) 5, be coated with goat-anti Mouse IgG polyclonal antibody on the control line (C line) 7.Test philosophy is: during detection, the anti-gp17 monoclonal antibody of colloid gold label of wrapping quilt on D29 bacteriophage in the nutrient solution and the test pen in advance is combined into bond, this bond is along with the capillarity of film is upwards moved arrival p-wire (T line) 5, react with the anti-gp23 monoclonal antibody on the film, a red stripes occurs.No matter whether have the D29 bacteriophage in the sample, when liquid level continued to migrate to fixedly sheep anti-mouse igg district band, a red stripes must appear in control line (C line) 7.If red stripes appears in p-wire (T line) 5, then point out positive findings, show the D29 bacteriophage that has capacity in the nutrient solution.Have only control line (C line) 7 red stripes to occur, then point out negative findings, show and do not deposit the D29 bacteriophage in the nutrient solution; Perhaps the D29 bacteriophage of Cun Zaiing does not reach the detection lower limit of test pen.
Compare with prior art, the present invention has following beneficial effect:
Fast inadequately based on existing bacteriophage rapid amplifying method, the defective that serological diagnostic method medium sensitivity and specificity are not high, the present invention is on the basis of bacteriophage rapid amplifying method, in conjunction with monoclonal antibody technique and colloidal gold method immunochromatography technique, designed D29 bacteriophage quick detection kit, diagnostic procedure was tapered in 30 hours, just can provide diagnostic result in second day that goes to a doctor in the patient; And having improved sensitivity and specificity, can distinguish tuberculosis and the outer tuberculosis of lung in the lung, is dead bacterium or viable bacteria in patient's sample.The present invention also provides a kind of phthisical special, sensitive rapid identification method, makes a definite diagnosis phthisical needs fast to satisfy, so that to the timely medication treatment of patient.Authentication method of the present invention can be distinguished tuberculosis and the outer tuberculosis of lung in the lung, and can distinguish dead bacterium and viable bacteria; In addition, the high specificity of test pen in the kit, susceptibility height, easy and simple to handle, the result is distinct, can be used for the pulmonary bacillus thalline Rapid identification of living in the sputum sample; More more importantly be that whole test is no more than 30 hours and just can finishes, just can provide diagnostic result, and can satisfy and make a definite diagnosis phthisical needs fast, in second day that goes to a doctor in the patient so that the timely medication of patient is given treatment to.
Description of drawings
Fig. 1 is the structural representation of biological test chip in the kit of fast detecting tubercle bacillus of the present invention;
Fig. 2 is the structural representation of the kit pnagus medius colloidal gold method immune chromatograph testing pen of fast detecting tubercle bacillus of the present invention.
In Fig. 1 and Fig. 2,1-working of plastics cap; 2-absorbs water excellent; The 3-sample pad; 4-collaurum pad; 5-p-wire (T line); 6-NC film (nitrocellulose filter); 7-control line (C line); The 8-absorbent filter; 9-working of plastics lower cover; The 10-drying agent; 11-working of plastics loam cake; The 12-watch window; The 13-PVC base plate.
Embodiment
The invention will be further elaborated with contrast test by the following examples:
Embodiment 1
One, MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation
1. plasmid construction: make up albumen gp17 (corresponding gene number: NC_046832) and gp23 (corresponding gene number: carrier for expression of eukaryon NC_046839) and prokaryotic expression carrier.Carrier for expression of eukaryon is selected plasmid pcDNA3.1/myc-His (-) A, and prokaryotic expression carrier is selected plasmid pET22b (+), through PCR, gel reclaims, enzyme is cut, and purifying connects, transform, behind the bacterium colony PCR, select positive colony, check order behind the extraction plasmid, obtain the correct clone gp17-2-2 (pET22b) of order-checking, gp23-1-3 (pcDNA3.1A).
2. expression of recombinant proteins and purifying
Change plasmid gp17-2-2 (pET22b) and gp23-1-3 (pcDNA3.1A) over to e. coli bl21, according to the normal experiment abduction delivering, 30 degrees centigrade of inducing temperatures, IPTG concentration 0.8mM.Obtain separately to distinguish purifying again behind the abduction delivering collection of illustrative plates, preparation contains the recombinant protein of His.
3. with respectively immune 6 mouse of resulting recombinant protein, the ELISA method detects serum titer, up to titre>100,000.
4. merge the SP2/0 mouse boosting cell, and screening stable cell line 10-20 strain.
5. select the highest a pair of cell line that can match again of affinity costant and optimize sandwich ELISA and reach best sensitivity, specificity, the range of linearity, repeatability.
6. select, go down to posterity and low-temperature storage totally 4 hybridoma cell strains.
7. Protein G post affinity purification mouse ascites or cell culture fluid obtain needed anti-gp17 monoclonal antibody and anti-gp23 monoclonal antibody.
Two, preparation D29 bacteriophage colloidal gold method immune chromatograph testing pen (this test pen structure is specifically seen Fig. 1 and Fig. 2)
1 basic demand
1.1 facility and quality of production management
" external diagnosis reagent production detailed rules for the implementation (trying) " requirement by State Food and Drug Administration's promulgation is implemented.
1.2 raw material and auxiliary material
Should meet existing " Chinese pharmacopoeia or " the main raw and auxiliary material quality control standard of Chinese biological goods " requirement.The chemical reagent of not including above-mentioned standard in should be not less than chemical pure.
2 special-purpose starting material
2.1 mouse-anti gp17 and anti-gp23 monoclonal antibody
Entrust exploitation of Ai Bimate biological medicine (Shanghai) Co., Ltd. and production, be affinity purification, purity>95%, affinity 〉=10 9, protein concentration 〉=2mg/ml.
2.2 sheep anti-mouse igg polyclonal antibody
Available from Xiamen Bo Sheng Bioisystech Co., Ltd, be affinity purification, purity>90%, protein concentration 〉=4mg/ml.
2.3 nitrocellulose membrane (NC film) 6 (see figure 2)s
Available from U.S. Millipore company, length standard: 100 ± 2m, width criteria: 2.54 ± 0.05cm, the time standard of metalling run out: 107~133 seconds.
2.4 collaurum pad 4 (seeing Fig. 1 and Fig. 2) (Glass Fiber Conjugate Pad)
Available from U.S. Millipore company, length standard: 50 ± 2m, width criteria: 8.0 ± 0.5mm, thickness calibration: 0.41 ± 0.05mm.
2.5 sample pad 3 and absorbent filter 8 (see figure 2)s
Available from Ahlstrom Filtration company, the wide 19 ± 0.5mm of sample pad, long 31 ± 0.2cm, the wide 20 ± 0.5mm of absorbent filter, long 31 ± 0.2cm.
Excellent 2 (see figure 2)s 2.6 absorb water
Available from U.S. Filtrona Fibertec company, length standard: 44.0 ± 0.6mm, width criteria: 11.88 ± 0.18mm, thickness calibration: 2.5 ± 0.2mm.
2.7 drying agent 10 (see figure 2)s
Available from Sud-Chemie company, thickness 0.125 ± 0.005inches, diameter 0.445 ± 0.005inches.
2.8PVC base plate 13 (see figure 1)s
Length standard: 31 ± 0.2cm, width criteria: 60.4~61.4mm.
2.9 working of plastics (comprise working of plastics cap 1, working of plastics lower cover 9 and working of plastics loam cake 11, see Fig. 2)
2.10HAuCl 4, trisodium citrate, available from Sigma company.
3 preparation procedures
3.1 the preparation of collaurum: adopt trisodium citrate reduction method to prepare collaurum, the colloid gold particle size is about 40nm.
The colloidal gold solution OD that makes answers>2.9, and the pH scope is 3.5~4.5.
3.2 the preparation of collaurum pad 4 (see figure 2)s
3.2.1 the dialysis of anti-gp17 monoclonal antibody: will resist the gp17 monoclonal antibody to place 1mM disodium phosphate soln dialysed overnight.
3.2.2 the preparation of anti-gp17 antibody-gold probe:
, under the condition of pH8.4,, after 30 minutes, under the condition of pH7.4, seal with the ratio of 1mg antibody/150OD collaurum, promptly obtain gp17 antibody-gold probe after centrifugal the concentrating with BSA with the anti-gp17 monoclonal antibody of colloid gold label.
This Au probe with collaurum film dilution (0.1M TAPS damping fluid, 20% sucrose, 3.75%BSA, pH8.0) be diluted to behind the OD2.0 standby.
3.2.3 preparation, the drying of anti-gp17 antibody colloidal gold pad:
Glass fibre after abundant the immersion, is taken out also to squeeze and removes unnecessary liquid in the gp17 of OD2.0 collaurum coating solution, be tiled on the special-purpose rack; In the environment of relative humidity≤20% drying at room temperature spend the night gp17 collaurum pad, standby.
3.3gp23 the preparation of nitrocellulose membrane (NC film)
3.3.1 the preparation of p-wire (T line) 5 (see figure 2)s spray membrane antibody solution:
10mM PBS solution with pH7.4 will resist the gp23 antibody dilution to 1.5mg/ml, and 0.22 μ m filters.
3.3.2 the preparation of control line (C line) 7 (see figure 2)s spray membrane antibody solution:
10mM PBS solution with pH7.4 is diluted to 2.0mg/ml with sheep anti-mouse antibody, and 0.22 μ m filters.
3.3.3gp23 preparation, the drying of nitrocellulose membrane (NC film):
Spray membrane antibody solution specking p-wire and control line on nitrocellulose filter with p-wire spray membrane antibody solution and control line respectively, and gp23 nitrocellulose filter drying at room temperature in the environment of relative humidity≤20% that preparation is finished is spent the night, standby.
3.4 the processing of sample pad 3 (seeing Fig. 1 and Fig. 2):
With untreated filter sample paper in filter sample paper treating fluid (50mM TAPS damping fluid fully soaks in pH8.0), be tiled on the special-purpose rack after taking out and extract unnecessary liquid, dried overnight under 37 ± 2 ℃ environment, standby.
3.5 the preparation of mucous membrane kilocalorie:
The shopwork envionmental humidity requires to be lower than 30% during mucous membrane.
As depicted in figs. 1 and 2, get one of 60mm * 300mm offset plate (end liner), paste on the center bag by after nitrocellulose membrane (NC film) 6,5 positions are down for the p-wire of NC film (T line); Paste the lower edge that a collaurum pad 4 and part cover nitrocellulose membrane (NC film) 6 at a side lower part of NC film p-wire (T line) 5, guarantee that both have that 1~2mm's is overlapping; Paste a sample pad of handling 3 and partly cover collaurum pad 4 lower edges at collaurum pad 4 downsides; Do not paste an end of sample pad at PVC base plate 13 and paste 8 one of absorbent filters, and guarantee that part covers NC film 6 upper limbs; To snap fits into polybag greatly after pasting, be lower than 20% low humidity in relative humidity and preserve between storing.
3.6 the cutting of biological test chip, assembling and packing
Shopwork envionmental humidity when cutting, assembling requires to be lower than 30%.
As shown in Figure 2, kilocalorie is cut into the biological test chip test paper bar of 6mm width with the Matrix2501 cutting machine, 6mm test strips, suction rod 2 and drying agent 10 are assembled into test pen by the test pen installation step working of plastics (comprising working of plastics cap 1, working of plastics lower cover 9 and working of plastics loam cake 11) of packing into according to the order of sequence, test pen is sealed with In Aluminium Foil Packing.
Three, the preparation of other component of D29 bacteriophage quick detection kit:
Specify: the plastic tube of all that mention in the In this Section 1~50ml, vial, plastic bottle all, could use after 30 minutes through 121 ℃ of high-temperature heat sterilizations.
1. sample preparation liquid (4% sodium hydroxide solution)
Take by weighing proper amount of sodium hydroxide, add purified water, be mixed with 4% sodium hydroxide solution, after filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, be sub-packed in the plastic bottle of 50ml (25ml/ bottle).
2. enrichment liquid
Michaelis 7H9 nutrient culture media 7g, lime chloride 2g, ampicillin 50g, amphotericin B 50g, bovine serum albumin(BSA) 200g, glucose 15g, oleic acid 2g and hydrogen peroxidase 0.2g all are dissolved in the pure water of 1000g and are prepared from; After filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, be sub-packed in the plastic bottle of 50ml (40ml/ bottle).
3. mycobacterium D29 bacteriophage
After the mycobacterium D29 bacteriophage that amplification is obtained is demarcated with the plaque count method, be diluted to 10 with Michaelis 7H9 fluid nutrient medium 9The concentration of individual/ml after filtering with the 0.22um bacterial filter, is sub-packed in local 100 grades Biohazard Safety Equipment in the plastic bottle of 2ml (1.2ml/ bottle).
4. agent for killing
Ferrous sulphate ammonia is mixed with 8% solution, after filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, is sub-packed in the plastic bottle of 1ml (1ml/ bottle).
5. mycobacterium smegmatis
After the mycobacterium smegmatis that amplification is obtained was demarcated concentration with spectrophotometer, (the thalline number was about 4 * 10 to be diluted to the concentration of 5mg/ml with Michaelis 7H9 fluid nutrient medium (through 121 ℃ of high-temperature heat sterilizations 30 minutes) 6Individual/ml, OD=1.0 during λ=600nm); In local 100 grades Biohazard Safety Equipment, be sub-packed in the plastic bottle of 10ml (10ml/ bottle).
Four, the packing of D29 bacteriophage quick detection kit (5 person-portions/box)
1. sample preparation liquid is 4% sodium hydroxide solution, 25ml/ bottle, totally one bottle;
2. enrichment liquid is Michaelis 7H9 liquid nutrient media, 40ml/ bottle, totally one bottle;
3. mycobacterium D29 bacteriophage, concentration 10 9Individual/ml, 1.2ml/ bottle, totally one bottle;
4. agent for killing is 8% ferrous sulphate ammonia solution, 1ml/ bottle, totally one bottle;
5. mycobacterium smegmatis, concentration 5mg/ml (the thalline number is about 4 * 10 6Individual/ml, 0D=1.0); The 10ml/ bottle, totally one bottle;
6. bacteriophage colloidal gold method immune chromatograph testing pen, 5 person-portions;
7. specimen collection bottle, the empty bottle of 10ml, totally 5 bottles.
Five, detect step
During detection, carry out according to the following steps:
1. (in sputum: the ratio of sample preparation liquid=1: 4) room temperature treatment sample to be checked is 20 minutes, the centrifugal supernatant that goes with sample preparation liquid;
2. get enrichment liquid 1~2ml, 37 ℃ increased bacterium 18~24 hours;
3. get the enrichment liquid 0.9ml that increases behind the bacterium, add concentration and be about 10 9The D29 mycobacteriophage 0.1ml of individual/ml, 37 ℃ of incubations 2 hours infect the pulmonary bacillus thalline;
4. add agent for killing 0.1ml in above-mentioned solution, all did not invade the bacteriophage of tubercle bacillus deactivation with aborting infection in 10 minutes in the room temperature effect;
5. add the enrichment liquid of 5ml in the above-mentioned solution once more, and add the mycobacterium smegmatis of 1ml, 37 ℃ of incubations 4 hours;
6. get the liquid to be measured of step e gained, detect result of determination with bacteriophage colloidal gold method immune chromatograph testing pen.
Six, interpretation as a result:
If red stripes appears in p-wire (T line), then point out positive findings, show the D29 that has capacity in the nutrient solution.Have only control line (C line) red stripes to occur, then point out negative findings, show not have D29 in the nutrient solution; Perhaps the D29 of Cun Zaiing does not reach the detection lower limit of test pen.
Embodiment 2
Remove following step, other step 1, two, five, six is with embodiment 1.
Three, the preparation of other component of D29 bacteriophage quick detection kit:
1. sample preparation liquid (2% sodium hydroxide solution)
Take by weighing proper amount of sodium hydroxide, add purified water, be mixed with 2% sodium hydroxide solution, after filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, be sub-packed in the plastic bottle of 50ml (25ml/ bottle).
2. enrichment liquid
Michaelis 7H9 nutrient culture media 5g, lime chloride 1g, ampicillin 25g, amphotericin B 25g, bovine serum albumin(BSA) 100g, glucose 10g, oleic acid 1g and hydrogen peroxidase 0.1g all are dissolved in the pure water of 1000g and are prepared from; After filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, be sub-packed in the plastic bottle of 50ml (40ml/ bottle).
3. mycobacterium D29 bacteriophage
After the mycobacterium D29 bacteriophage that amplification is obtained is demarcated with the plaque count method, be diluted to 10 with Michaelis 7H9 fluid nutrient medium 9The concentration of individual/ml after filtering with the 0.22um bacterial filter, is sub-packed in local 100 grades Biohazard Safety Equipment in the plastic bottle of 2ml (1.2ml/ bottle).
4. agent for killing
Ferrous sulphate ammonia is mixed with 5% solution, after filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, is sub-packed in the plastic bottle of 1ml (1ml/ bottle).
5. mycobacterium smegmatis
After the mycobacterium smegmatis that amplification is obtained was demarcated concentration with spectrophotometer, (the thalline number was about 2.4 * 10 to be diluted to the concentration of 3mg/ml with Michaelis 7H9 fluid nutrient medium (through 121 ℃ of high-temperature heat sterilizations 30 minutes) 6Individual/ml, OD=0.6 during λ=600nm); In local 100 grades Biohazard Safety Equipment, be sub-packed in the plastic bottle of 10ml (10ml/ bottle).
Four, the packing of D29 bacteriophage quick detection kit (5 person-portions/box)
1. sample preparation liquid is 2% sodium hydroxide solution, 25ml/ bottle, totally one bottle;
2. enrichment liquid is Michaelis 7H9 liquid nutrient media, 40ml/ bottle, totally one bottle;
3. mycobacterium D29 bacteriophage, concentration 10 9Individual/ml, 1.2ml/ bottle, totally one bottle;
4. agent for killing is 5% ferrous sulphate ammonia solution, 1ml/ bottle, totally one bottle;
5. mycobacterium smegmatis, concentration 3mg/ml (the thalline number is about 2.4 * 10 6Individual/ml, OD=0.6); The 10ml/ bottle, totally one bottle;
6. bacteriophage colloidal gold method immune chromatograph testing pen, 5 person-portions;
7. specimen collection bottle, the empty bottle of 10ml, totally 5 bottles.
Embodiment 3
Remove following step, other step 1, two, five, six is with embodiment 1.
Three, the preparation of other component of D29 bacteriophage quick detection kit:
1. sample preparation liquid (3% sodium hydroxide solution)
Take by weighing proper amount of sodium hydroxide, add purified water, be mixed with 3% sodium hydroxide solution, after filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, be sub-packed in the plastic bottle of 50ml (25ml/ bottle).
2. enrichment liquid
Michaelis 7H9 nutrient culture media 6g, lime chloride 1.5g, ampicillin 37.5g, amphotericin B 37.5g, bovine serum albumin(BSA) 150g, glucose 12.5g, oleic acid 1.5g and hydrogen peroxidase 0.15g all are dissolved in the pure water of 1000g and are prepared from; After filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, be sub-packed in the plastic bottle of 50ml (40ml/ bottle).
3. mycobacterium D29 bacteriophage
After the mycobacterium D29 bacteriophage that amplification is obtained is demarcated with the plaque count method, be diluted to 10 with Michaelis 7H9 fluid nutrient medium 9The concentration of individual/ml after filtering with the 0.22um bacterial filter, is sub-packed in local 100 grades Biohazard Safety Equipment in the plastic bottle of 2ml (1.2ml/ bottle).
4. agent for killing
Ferrous sulphate ammonia is mixed with 6% solution, after filtering with the 0.22um bacterial filter, in local 100 grades superclean bench, is sub-packed in the plastic bottle of 1ml (1ml/ bottle).
5. mycobacterium smegmatis
After the mycobacterium smegmatis that amplification is obtained was demarcated concentration with spectrophotometer, (the thalline number was about 3.2 * 10 to be diluted to the concentration of 4mg/ml with Michaelis 7H9 fluid nutrient medium (through 121 ℃ of high-temperature heat sterilizations 30 minutes) 6Individual/ml, OD=0.8 during λ=600nm); In local 100 grades Biohazard Safety Equipment, be sub-packed in the plastic bottle of 10ml (10ml/ bottle).
Four, the packing of D29 bacteriophage quick detection kit (5 person-portions/box)
1. sample preparation liquid is 3% sodium hydroxide solution, 25ml/ bottle, totally one bottle;
2. enrichment liquid is Michaelis 7H9 liquid nutrient media, 40ml/ bottle, totally one bottle;
3. mycobacterium D29 bacteriophage, concentration 10 9Individual/ml, 1.2ml/ bottle, totally one bottle;
4. agent for killing is 6% ferrous sulphate ammonia solution, 1ml/ bottle, totally one bottle;
5. mycobacterium smegmatis, concentration 4mg/ml (the thalline number is about 3.2 * 10 6Individual/ml, OD=0.8); The 10ml/ bottle, totally one bottle;
6. bacteriophage colloidal gold method immune chromatograph testing pen, 5 person-portions;
7. specimen collection bottle, the empty bottle of 10ml, totally 5 bottles.
Contrast test:
This contrast test is sample with the sputum, adopts sputum cultivation, phage splitting method and the inventive method to carry out the detection of tubercle bacillus respectively, testing result as shown in Table 1 and Table 2:
Table 1
Method/kit title Technical characterictic Kit producer Detect sample Testing result
A: sputum cultivation Solid agar double dish is cultivated tubercle bacillus ??- 9 routine samples 4 examples are positive; 5 examples are negative;
B: phage splitting method Bacteriophage is infected bacteriophage+solid agar double dish that bacteriophage+MTB has been invaded in indicator cells amplification that tubercle bacillus (MTB)+kill is not invaded MTB, observes plaque Britain BIOTEC company tubercle bacillus detection kit (FAST Plaque TB TM) 9 routine samples 4 examples are positive; 5 examples are negative;
C: the present invention Bacteriophage is infected bacteriophage+bacteriophage colloidal gold method immune chromatograph testing pen that bacteriophage+MTB has been invaded in the amplification of shame dirt bacillus that tubercle bacillus (MTB)+kill is not invaded MTB Inverness Medical Shanghai Co., Ltd. 9 routine samples 4 examples are positive; 5 examples are negative;
Table 2
Conclusion: from table 1 and table 2, this test has detected the positive and 5 routine negative sputum samples of 4 examples altogether; Adopt the inventive method to compare with adopting sputum cultivation, phage splitting method, it is consuming time generally in 30 hours to adopt the inventive method to detect, to obviously be less than the detection time of adopting the sputum cultivation and adopting the phage splitting method, in addition, adopt the inventive method to compare with adopting other 2 kinds of detection methods, its testing result is in full accord, promptly adopts the inventive method can detect tubercle bacillus quickly and accurately.

Claims (5)

1, the method for a kind of fast detecting tubercle bacillus is characterized in that, comprises the steps: the first step, uses the phage-infect tubercle bacillus; In second step, kill tubercle bacillus and do not infect bacteriophage outward; The 3rd step, the bacteriophage that amplification has been infected; In the 4th step, with phage-resistant antibody standard gold after, thereby adopt the existence of fast immune chromatographic method detection bacteriophage whether in the judgement sample whether tubercle bacillus to be arranged.
2, the method for fast detecting tubercle bacillus according to claim 1, it is characterized in that the 4th the step specifically comprise:
(1). prepare special monoclonal antibody at bacteriophage;
(2). special monoclonal antibody is attached to the colloid gold particle of activation;
(3). preparation bacteriophage colloidal gold method immune chromatograph testing pen, p-wire and control line are set on test-strips, with the control detection quality, detect with bacteriophage colloidal gold method immune chromatograph testing pen, whether tubercle bacillus is arranged in the judgement sample.
3, the method for fast detecting tubercle bacillus according to claim 1, it is characterized in that, the 3rd step adopted the liquid cultivating method amplification to infect the bacteriophage of tubercle bacillus, adopts Michaelis 7H9 liquid nutrient media and add the amplification host of shame dirt bacillus as bacteriophage in amplification procedure.
4, the kit of a kind of fast detecting tubercle bacillus is characterized in that, comprises sample preparation liquid, enrichment liquid, agent for killing, mycobacterium smegmatis, mycobacteriophage, bacteriophage colloidal gold method immune chromatograph testing pen;
Described sample preparation liquid is 2~4% sodium hydroxide solution; Described enrichment liquid is by Michaelis 7H9 nutrient culture media 5-7 weight portion, lime chloride 1-2 weight portion, ampicillin 25-50 weight portion, amphotericin B 25-50 weight portion, bovine serum albumin(BSA) 100-200 weight portion, glucose 10-15 weight portion, oleic acid 1-2 weight portion and hydrogen peroxidase 0.1-0.2 weight portion, all is dissolved in the pure water of 1000 weight portions being prepared from; Described agent for killing is 5~8% ferrous sulphate ammonia solution; Described mycobacterium smegmatis is the 3~5mg/ml mycobacterium smegmatis that is dissolved in Michaelis 7H9 fluid nutrient medium; Described mycobacteriophage is the mycobacterium D29 bacteriophage that is dissolved in Michaelis 7H9 fluid nutrient medium; After described bacteriophage colloidal gold method immune chromatograph testing pen was put into the plastic housing closure by the biological test chip, the aluminium foil bag of putting into a suitable size again was sealed to form.
5, the kit of fast detecting tubercle bacillus according to claim 4 is characterized in that, described biological test chip comprises PVC base plate, sample pad, collaurum pad, nitrocellulose filter, absorbent filter; Be coated with the compound of special a kind of monoclonal antibody-collaurum at the D29 bacteriophage above the described collaurum pad, be coated with p-wire and control line above the described nitrocellulose filter, be coated with special another kind of monoclonal antibody on this p-wire, be coated with goat-anti Mouse IgG polyclonal antibody on this control line at the D29 bacteriophage.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011020434A1 (en) * 2009-08-20 2011-02-24 上海英伯肯医学生物技术有限公司 Antibody binding to the protein of the bacteriophage of mycobacterium tuberculosis, and the preparation method and use thereof
CN102507945A (en) * 2011-12-05 2012-06-20 河北省科学院生物研究所 Sulfamethazine enzyme-linked immunoassay kit
CN104034889A (en) * 2014-06-12 2014-09-10 江崇才 Method for detecting mycobacterium tuberculosis
CN104970769A (en) * 2015-06-09 2015-10-14 黄义超 Tuberculosis category diagnosis device
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CN1425775A (en) * 2001-12-11 2003-06-25 李克生 Tuberculosis antibody gold label test strip and its preparing method
CN100494352C (en) * 2006-12-30 2009-06-03 华中农业大学 Immune colloidal gold test paper strip for detecting bovine tuberculosis antibody and its preparation method

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011020434A1 (en) * 2009-08-20 2011-02-24 上海英伯肯医学生物技术有限公司 Antibody binding to the protein of the bacteriophage of mycobacterium tuberculosis, and the preparation method and use thereof
CN102507945A (en) * 2011-12-05 2012-06-20 河北省科学院生物研究所 Sulfamethazine enzyme-linked immunoassay kit
CN104034889A (en) * 2014-06-12 2014-09-10 江崇才 Method for detecting mycobacterium tuberculosis
CN104970769A (en) * 2015-06-09 2015-10-14 黄义超 Tuberculosis category diagnosis device
WO2019018886A1 (en) * 2017-07-23 2019-01-31 Future Biosolutions Pty Ltd Method for rapid detection and enumeration of viruses, bacteriophage and/or bacteria

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