CN107641643A - A kind of method of β hemolytic streptococcus in quick detection textile - Google Patents
A kind of method of β hemolytic streptococcus in quick detection textile Download PDFInfo
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Abstract
The invention discloses in a kind of quick detection textileβ‑The method of hemolytic streptococcus, with reference to two kinds of technologies of IMS and RPA, using in immunomagnetic ca pture technology enriched sampleβ‑Hemolytic streptococcus, forβ‑The specificity of type hemolytic streptococcusHtrATarget gene, design a set of specific primer and probe using RPA technologies as core technology, be enriched with RPA technology for detectionβ‑Type hemolytic streptococcus.This method whole process need not cultivate, and save time, quick, efficiently, specific good, high sensitivity, strong interference immunity is applied widely, and testing cost is low, is applicable not only to health and epidemic prevention and health supervision department, applies also for the detection of enterprise and domestic hygiene.
Description
Technical field
The present invention relates to a kind of detection method of beta hemolytic streptococcus, and in particular to and β in a kind of quick detection textile-
The method of hemolytic streptococcus.
Background technology
Beta hemolytic streptococcus (β-hemolytic streptococcus) is distributed more widely in nature, be present in water,
In air, dust, the oral cavity of excrement and healthy humans and animals, nasal cavity, throat.Pacify in streptococcus with environmental sanitation and the person
Complete relevant streptococcus is mainly beta hemolytic streptococcus, by its C antigen, can be divided into 18 groups, and what people was caused a disease belongs to A mostly
Group, B groups, C groups and G groups.Beta hemolytic streptococcus is stronger to virulence, the invasiveness of people, can produce a variety of ectoenzymes and exotoxin,
Cause various purulent diseases, pneumonia, wound infection and septicemia etc..Can by directly contacting, the air spittle or skin, mucous membrane
Wound infection is propagated, and generally causes purulent inflammation and the respiratory tract infection of skin and hypodermis, can also cause scarlet fever, stream
Row pharyngitis, acute tonsillitis and impetigo, or even serious invasive infection such as septicemia, necrotizing fasciitis can be caused
With streptococcus toxic shock syndrome.
Beta hemolytic streptococcus category gram-positive cocci, no brood cell's atrichia, canescence, half are formed after blood plate culture
It is transparent, surface is smooth, neat in edge, diameter be in 0.5mm~0.75mm tiny bacterium colony, surrounding formed a 2mm~4mm it is wide,
Clear-cut, also referred to as fully transparent colourless zone of hemolysis, beta hemolysis.
The bacterium resistance is not general strong, and 60 DEG C of 30min are killed, sensitive to common disinfectants, but resist drying ability is strong,
It can be survived the several months in dust is dried.Due to particularity existing for textile (exposure is in atmosphere), it is easy to pollution is attached to,
It is communicated by contact to the mankind.
The bacterium laboratory cultures nutritional requirement is higher, aerobic or facultative anaerobic bacteria, undergrowth on ordinary culture medium, needs to mend
Congested clear, blood, ascites, most of bacterial strains need the growth factors such as riboflavin and vitamin B6, nicotinic acid, therefore are unfavorable for traditional training
Support.Optimum growth temperature is 37 DEG C, can be grown at 20 DEG C~42 DEG C, optimal pH 7.4-7.6.Easily grow up in serum broth
Chain, ttom of pipe grow in cotton-shaped or granular precipitate.Decomposition glucose, the sour not aerogenesis of production, to lactose, mannitol, salicin, sorb
Alcohol, gossypose, gill fungus sugar, the capacity of decomposition of aesculin are because of different strains and different.Synanthrin is not typically decomposed, is not dissolved by bile, is touched
Enzyme is negative.
At present, for beta hemolytic streptococcus detection method mainly have that FDA recommends be separately cultured, biochemical and serology is reflected
Fixed, immunological method and PCR.Traditional detection method program is cumbersome, cycle length, it usually needs more than 6d, beta hemolytic streptococcus
Belong to difficult culture microorganism in conventional detection, false negative often occurs in the relatively low conventional method of sensitivity；Immunological method prepares antibody
It is more difficult, easily there is cross pollution, specificity and sensitivity are relatively low；PCR methods are sensitive, accurate, quick, alternative traditional detection
Method, but need expensive instrument and equipment, the technical requirements higher to testing staff, it is difficult to popularized in grass-roots unit.
Immunomagnetic beads beneficiation technologies (immunomagnetic separation, IMS)：Magnetic is coated on target bacteria antibody
Bead surface, the objective microbe in sample liquid is then adsorbed under the influence of a magnetic field, makes itself and influence and Interference Detection sensitivity
Sample residue and other miscellaneous bacterias separate, play a part of concentration, purification of target microorganism, so as to improve sensitivity and detection
Rate.IMS methods are easy to operate, immunomagnetic beads need to be only added in sample liquid, after shaken at room temperature mixes the regular hour, in magnetic field
Washed repeatedly with buffer solution under effect, the immunomagnetic beads for capturing object bacteria does not have to separate with object bacteria, directly carries out follow-up
Experiment.Fast with separating rate, efficiency high, favorable repeatability is simple to operate, it is not necessary to expensive instrument and equipment, does not influence quilt
The advantages that separating the biological character and function of cell or other biomaterials, have in terms of microorganism detection very big excellent
Gesture.
Recombinase polymerase isothermal amplification technique (recombinase ploymerase amplification, RPA)：It is
A kind of new isothermal amplification technology, the technology utilize recombinase and single-stranded knot using T4 bacteriophage nucleic acids replicanism as principle
The hop protein specific bond of cooperative achievement primer and template at normal temperatures.Amplified production can be surveyed by agarose electrophoresis, clone
The methods of sequence or real-time fluorescence, is detected.Wherein adding oligonucleotide probe, monitoring can improve the specificity of method in real time, be applied to
The quick detection of pathogenic microorganism.
Compared with other DNA detection methods, real-time fluorescence RPA methods have the advantage that：1. core is carried out under constant temperature
The amplification of acid, has broken away from the limitation of the accurate thermal cycle of PCR courses of reaction.2. need not higher or accurate temperature, and can be
Reacted between 25 DEG C~42 DEG C.3. reaction speed is very fast, 15min~30min is only typically needed from starting to completing, far faster than
Other isothermal amplification techniques or round pcr.4. high sensitivity and specificity, its sensitivity and specificity with real-time fluorescence PCR without
Difference.5. it is simple to operate, a variety of instruments (portable small-sized real-time fluorescence detecting instrument, real-time fluorescence quantitative PCR instrument can be used
Device) detected, wherein using portable small-sized real-time fluorescence detecting instrument, it is cheap, be advantageous at grass-roots unit scene
Examinations and popularization and application.
The culture-based method qualitative detection that the detection of field of textiles beta hemolytic streptococcus all uses at present, detected in method
It is also all roughly the same in program, increase bacterium → Selective Separation → microscopy and biochemical test, it is necessary to more than 6d.Textile samples composition
Complexity, traditional detection method were readily incorporated multiple error, caused false negative result, therefore, will according to the characteristic of textile sample
IMS and RPA technological sciences combine, and establish a kind of specific enrichment, quick, easy, accurate detection method, are beta hemolysis hammer
The detection of bacterium provides new developing direction, is expected to turn into easy conventional detection means, be particularly suitable for use in basic unit's inspection and quarantine
Mechanism, it is important for improving product health level, ensureing common people's personal safety and promoting textile development of international trade to have
Meaning.
The content of the invention
In view of the shortcomings of the prior art, the invention provides a kind of side of beta hemolytic streptococcus in quick detection textile
Method, specific enrichment, quick, easy, accurate effect are reached.Concrete technical scheme is as follows：
The method of beta hemolytic streptococcus, comprises the following steps in a kind of quick detection textile：
Step 1: the preparation of immunomagnetic beads
0.1mL magnetic beads liquid is taken in the centrifuge tube for filling 1.0mL PBST buffer solutions, is mixed, 3000r/min centrifugation 2min,
Abandon liquid；Repeat the above steps cleaning 3 times, and adding 1.0mL PBST buffer solutions makes magnetic bead be resuspended wherein；Into above-mentioned re-suspension liquid
0.1mL beta hemolytic streptococcus immune serums are added, put shaking table, the light and slow rotational oscillation 3h of room temperature；Cleaned with 1 × washing lotions of 1.0mL
After 5 times, 0.5mL re-suspension liquids are added, are saved backup in 4 DEG C；
Step 2: RPA primers and probe design
Coding HtrA target-gene sequence is downloaded in ncbi database, sequence alignment is carried out using CLUSTAL X softwares,
One section is have found in HtrA genes only to guard in the specific sequence of A, B, C, G group of beta hemolytic streptococcus, and in this sequence
A set of specific primer and probe using RPA technologies as core technology is designed in row；
Step 3: the immunomagnetic beads enrichment of beta hemolytic streptococcus
Sterile working is weighed in 25g samples to the sterile homogenizing bag of band filter screen equipped with 100mL peptone waters, light and slow vibration
30min；Sample is filtered, supernatant is shifted in centrifuge tube, 5000r/min centrifugation 20min, abandons supernatant fluid, precipitation is resuspended in
In 1.0mL PBST buffer solutions；Whole sample suspensions are taken in the tubule equipped with 0.1mL immunomagnetic beads suspensions, the light and slow rotation of room temperature
Vibrate 30min；Liquid in pipe is carefully removed from, 1.0mL washing lotions is added and mixes, washing lotion is removed after 3min, repeated washing 5 times, is added
0.1mL re-suspension liquids make the magnetic bead of absorption beta hemolytic streptococcus be resuspended wherein；Suspension containing magnetic beads with beta hemolytic streptococcus can
It is directly used in extraction DNA of bacteria；
Step 4: RPA is identified
4.1) preparation of DNA profiling
100 μ L beta hemolytic streptococcuses suspension containing magnetic beads are taken in 0.5mL pipes, 100 DEG C of water-bath 10min, 5000r/min centrifugations
5min, supernatant is taken, -20 DEG C save backup；
4.2) RPA amplified reactions
4.2.1) RPA reaction tube numbers n (the pipe negative controls of n=1 pipes blank control+1 according to required for being set sample size
+ 1 pipe positive control+sample number)；Table is sequentially added into the 0.2mL TwistAmp Exo RPA reaction tubes containing lyophilized enzyme powder
Each composition in 1,2000r/min centrifugations 10sec, RPA portable fluorescence detectors are placed on by reaction tube after fully mixing
(Twista) real-time fluorescence amplification, 37 DEG C of isothermal reaction 20min are carried out in；
The reaction system of table 1
| RPA reagents | 1 pipe tests dosage | N pipes test dosage |
| Rehydration buffer | 29.5μL | 29.5×nμL |
| RPA-F | 2.0μL | 2.0×nμL |
| RPA-R | 2.0μL | 2.0×nμL |
| RPA-P | 0.5μL | 0.5×nμL |
| Deionized water | 14.0μL | 14.0×nμL |
| DNA profiling | 2.0μL | 2.0×nμL |
| Cumulative volume | 50.0μL | 50.0×nμL |
4.2.2 blank control, negative control, positive control) are set
Negative control, blank control and positive control are respectively provided with per secondary response；
Blank control：DNA profiling is replaced with water；
Negative control：Sample, the template that extraction DNA reacts as RPA are replaced with water；
Positive control：The template that beta hemolytic streptococcus reference culture extraction DNA reacts as RPA；
4.3) result is observed
After reaction terminates, the amplification curve of observation RPA portable fluorescence detectors (Twista) generation, blank control, the moon
Property control without amplification curve, positive control has a typical S types amplification curve, judges that experiment is effective, and otherwise experiment is considered as invalid；
Step 5: result is reported
In blank control, negative control without amplification curve, on the premise of positive control has typical S types amplification curve, sample
Reported without amplification curve in sample and do not detect beta hemolytic streptococcus；There are the S type amplification curve reports of similar positive control in sample
Accuse in sample and detect beta hemolytic streptococcus.
The equipment and material used in the present invention are as follows：
1 equipment and material
The basic equipment and material of 1.1 conventional microbiological detections.
1.2 sterile scissors, tweezers.
1.3 electronic balance：0.01g.
1.4 sterile homogenizing bags (band filter screen).
1.5 slap type homogenizer：400mL.
1.6 shaking table.
1.7 micropipettor：The μ L of μ L of range 1~20；The μ L of μ L of range 200~1000.
1.8 sterilizing Eppendorf centrifuge tubes：1.5mL.
1.9 supercentrifuge：2000r/min~13000r/min.
1.10 portable fluorescence detectors (Twista).
2 culture mediums and reagent
2.1 peptone waters (are purchased from overpass company).
2.2 beta hemolytic streptococcus immune serums (デ ソ カ Sheng Yan Co., Ltd. product).
2.3 magnetic bead liquid DynabeadsM_280Sheep anti_Rabbit IgG (Dynal Products).
2.4PBST buffer solutions (Dynal Products).
2.5 10 × washing lotion (Dynal Products).
2.6 re-suspension liquids (Dynal Products).
2.7 sterilizing distilled waters.
2.8RPA fluorescence detection reagent kits (TwistAmp DNA Amplification Exo Kits) (Britain TwistDX
Products).
2.9 primer：RPA is 30~35 nucleotides to primer length requirement, according to the HtrA bases of beta hemolytic streptococcus
Because of conserved sequence (GenBank accession number：KB904189.1), a probe is devised, 1 upstream is devised in the both sides of probe
Primer and 1 anti-sense primer.Primer is synthesized by Nanjing Genscript Biotechnology Co., Ltd..
Table 2RPA primer and probes
3 methods detect program
Method detection program is shown in Fig. 1.
4 prevent the measure of pollution and offal treatment
Before and after 4.1 detections, use for laboratory disinfection by ultraviolet light and by cleaning, wiping the various utensils of removal and equipment list repeatedly
The DNA of face residual.
Strict partition operates in 4.2 detection process, need to meet GB/T 27403《Good Laboratory controls specification food molecule
Biological Detection》It is required that to prevent the cross pollution of nucleic acid.
4.3 detection discarded objects need to meet GB 19489《Laboratory biosafety General Requirement》It is required that through autoclaving etc.
Harmless treatment.
This method combination two kinds of technologies of IMS and RPA, using beta hemolysis hammer in immunomagnetic ca pture technology enriched sample
Bacterium；Research shows that hemolytic streptococcal infection human host is relevant with the C5a- peptases of HtrA gene codes, and this gene is not
There is very high uniformity in same pathogenic hemolytic streptococcus, but be not present in other bacterial strains；Existing mouse experiment model
The virulence of display HtrA deletion mutation strains substantially weakens, and has C5a peptases at present and plays protective immunity to A, B, C, G group of streptococcus
The patent research of effect, thus for beta hemolytic streptococcus specific HtrA target gene, design it is a set of using RPA technologies as
The specific primer and probe of core technology, the beta hemolytic streptococcus being enriched with RPA technology for detection, establish for weaving
The IMS- real-time fluorescence RPA detection methods of the beta hemolytic streptococcus rapid sensitive of product.
The method of beta hemolytic streptococcus in IMS- real-time fluorescences RPA quick detection textiles provided by the invention, with showing
There is technology to compare to have the advantages that：
1. this method is easy to be quick, IMS- real-time fluorescence RPA methods detection beta hemolytic streptococcus can be completed in 1d, other
Detection method negative findings need 2d~5d, positive findings needs more than 6d；
2. this method specificity is good, the specificity of inter-species is individually lacked with immunomagnetic beads enrichment of bacterial, cross reaction eliminates
Not exclusively, it is possible to and non-specific binding occurs for other miscellaneous bacterias；Real-time fluorescence is utilized on the basis of immunomagnetic beads enrichment
RPA is identified that method has bispecific in itself, and selectivity is higher, result is more accurate；
3. this method susceptibility is high, susceptibility is the whether ripe effective important indicator of the method for inspection, IMS- real-time fluorescences
RPA methods detection lower bound streptococcic to beta hemolysis is 10CFU/mL, has met or exceeded the detection of other detection methods
Lower bound；
4. this method strong interference immunity, target bacteria is directly captured from sample using immunomagnetic beads method, eliminates DNA cloning
Inhibiting substances；Simultaneously because fluorescence RPA specific amplification beta hemolytic streptococcus DNA, other type haemolysis of immunomagnetic beads absorption
Property streptococcus without interference with RPA, IMS- real-time fluorescence RPA technologies solve interference and suppress problem；
5. this method combination two kinds of technical characterstics of IMS and RPA, applied widely, testing cost is low, is applicable not only to health
Epidemic prevention and health supervision department, apply also for the detection of enterprise and domestic hygiene；
6. this method combines two kinds of technical characterstics, the identification of beta hemolytic streptococcus is set simply to be divided into 2 steps：IMS technologies
Bacterium → RPA technical appraisement, whole process need not cultivate in enriched sample, save time, quick, efficiently, entirely test
Journey only needs a portable small-sized real-time fluorescence detector, and cost is cheap, and (portable small-sized real-time fluorescence detector only needs
Hundreds of yuan with regard to that can purchase), suitable basic unit's popularization and application, or even Site Detection can be carried out.
Brief description of the drawings
Fig. 1 is that method detects program.
Fig. 2 is specific test amplification curve diagram.
Fig. 3 is DNA concentration test result figure.
Fig. 4 is sensitivity test result figure, and wherein DNA profiling concentration 1 is 46.534ng/ μ L, and 2 be 4.6534ng/ μ L, 3
It is 0.046534ng/ μ L for 0.46534ng/ μ L, 4,5 be 0.0046534ng/ μ L.
Fig. 5 is 1~No. 20 sample RPA amplification curve diagram.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with being preferable to carry out for the present invention
Example, and coordinate accompanying drawing to be described in detail.
Embodiment 1
The method of beta hemolytic streptococcus, comprises the following steps in a kind of quick detection textile：
Step 1: the preparation of immunomagnetic beads
0.1mL magnetic beads liquid is taken in the centrifuge tube for filling 1.0mL PBST buffer solutions, is mixed, 3000r/min centrifugation 2min,
Abandon liquid；Repeat the above steps cleaning 3 times, and adding 1.0mL PBST buffer solutions makes magnetic bead be resuspended wherein；Into above-mentioned re-suspension liquid
0.1mL beta hemolytic streptococcus immune serums are added, put shaking table, the light and slow rotational oscillation 3h of room temperature；Cleaned with 1 × washing lotions of 1.0mL
After 5 times, 0.5mL re-suspension liquids are added, are saved backup in 4 DEG C；
Step 2: RPA primers and probe design
Coding HtrA target-gene sequence is downloaded in ncbi database, sequence alignment is carried out using CLUSTAL X softwares,
One section is have found in HtrA genes only to guard in the specific sequence of A, B, C, G group of beta hemolytic streptococcus, and in this sequence
A set of specific primer and probe using RPA technologies as core technology is designed in row；
Step 3: the immunomagnetic beads enrichment of beta hemolytic streptococcus
Sterile working is weighed in 25g samples to the sterile homogenizing bag of band filter screen equipped with 100mL peptone waters, light and slow vibration
30min；Sample is filtered, supernatant is shifted in centrifuge tube, 5000r/min centrifugation 20min, abandons supernatant fluid, precipitation is resuspended in
In 1.0mL PBST buffer solutions；Whole sample suspensions are taken in the tubule equipped with 0.1mL immunomagnetic beads suspensions, the light and slow rotation of room temperature
Vibrate 30min；Liquid in pipe is carefully removed from, 1.0mL washing lotions is added and mixes, washing lotion is removed after 3min, repeated washing 5 times, is added
0.1mL re-suspension liquids make the streptococcic magnetic bead of absorption beta hemolysis be resuspended wherein；Suspension containing magnetic beads with beta hemolytic streptococcus
It can be directly used for extracting DNA of bacteria；
Step 4: RPA is identified
4.1) preparation of DNA profiling
100 μ L beta hemolytic streptococcuses suspension containing magnetic beads are taken in 0.5mL pipes, 100 DEG C of water-bath 10min, 5000r/min centrifugations
5min, supernatant is taken, -20 DEG C save backup；
4.2) RPA amplified reactions
4.2.1) RPA reaction tube numbers n (the pipe negative controls of n=1 pipes blank control+1 according to required for being set sample size
+ 1 pipe positive control+sample number)；Table is sequentially added into the 0.2mL TwistAmp Exo RPA reaction tubes containing lyophilized enzyme powder
Each composition in 1,2000r/min centrifugations 10sec, RPA portable fluorescence detectors are placed on by reaction tube after fully mixing
(Twista) real-time fluorescence amplification, 37 DEG C of isothermal reaction 20min are carried out in；
The reaction system of table 1
| RPA reagents | 1 pipe tests dosage | N pipes test dosage |
| Rehydration buffer | 29.5μL | 29.5×nμL |
| RPA-F | 2.0μL | 2.0×nμL |
| RPA-R | 2.0μL | 2.0×nμL |
| RPA-P | 0.5μL | 0.5×nμL |
| Deionized water | 14.0μL | 14.0×nμL |
| DNA profiling | 2.0μL | 2.0×nμL |
| Cumulative volume | 50.0μL | 50.0×nμL |
4.2.2 blank control, negative control, positive control) are set
Negative control, blank control and positive control are respectively provided with per secondary response；
Blank control：DNA profiling is replaced with water；
Negative control：Sample, the template that extraction DNA reacts as RPA are replaced with water；
Positive control：The template that beta hemolytic streptococcus reference culture extraction DNA reacts as RPA；
4.3) result is observed
After reaction terminates, the amplification curve of observation RPA portable fluorescence detectors (Twista) generation, blank control, the moon
Property control without amplification curve, positive control has a typical S types amplification curve, judges that experiment is effective, and otherwise experiment is considered as invalid；
Step 5: result is reported
In blank control, negative control without amplification curve, on the premise of positive control has typical S types amplification curve, sample
Reported without amplification curve in sample and do not detect beta hemolytic streptococcus.
Embodiment 2
The method of beta hemolytic streptococcus, comprises the following steps in a kind of quick detection textile：
Step 1: the preparation of immunomagnetic beads
0.1mL magnetic beads liquid is taken in the centrifuge tube for filling 1.0mL PBST buffer solutions, is mixed, 3000r/min centrifugation 2min,
Abandon liquid；Repeat the above steps cleaning 3 times, and adding 1.0mL PBST buffer solutions makes magnetic bead be resuspended wherein；Into above-mentioned re-suspension liquid
0.1mL beta hemolytic streptococcus immune serums are added, put shaking table, the light and slow rotational oscillation 3h of room temperature；Cleaned with 1 × washing lotions of 1.0mL
After 5 times, 0.5mL re-suspension liquids are added, are saved backup in 4 DEG C；
Step 2: RPA primers and probe design
Coding HtrA target-gene sequence is downloaded in ncbi database, sequence alignment is carried out using CLUSTAL X softwares,
One section is have found in HtrA genes only to guard in the specific sequence of A, B, C, G group of beta hemolytic streptococcus, and in this sequence
A set of specific primer and probe using RPA technologies as core technology is designed in row；
Step 3: the immunomagnetic beads enrichment of beta hemolytic streptococcus
Sterile working is weighed in 25g samples to the sterile homogenizing bag of band filter screen equipped with 100mL peptone waters, light and slow vibration
30min；Sample is filtered, supernatant is shifted in centrifuge tube, 5000r/min centrifugation 20min, abandons supernatant fluid, precipitation is resuspended in
In 1.0mL PBST buffer solutions；Whole sample suspensions are taken in the tubule equipped with 0.1mL immunomagnetic beads suspensions, the light and slow rotation of room temperature
Vibrate 30min；Liquid in pipe is carefully removed from, 1.0mL washing lotions is added and mixes, washing lotion is removed after 3min, repeated washing 5 times, is added
0.1mL re-suspension liquids make the streptococcic magnetic bead of absorption beta hemolysis be resuspended wherein；Suspension containing magnetic beads with beta hemolytic streptococcus
It can be directly used for extracting DNA of bacteria；
Step 4: RPA is identified
4.1) preparation of DNA profiling
100 μ L beta hemolytic streptococcuses suspension containing magnetic beads are taken in 0.5mL pipes, 100 DEG C of water-bath 10min, 5000r/min centrifugations
5min, supernatant is taken, -20 DEG C save backup；
4.2) RPA amplified reactions
4.2.1) RPA reaction tube numbers n (the pipe negative controls of n=1 pipes blank control+1 according to required for being set sample size
+ 1 pipe positive control+sample number)；Table is sequentially added into the 0.2mL TwistAmp Exo RPA reaction tubes containing lyophilized enzyme powder
Each composition in 1,2000r/min centrifugations 10sec, RPA portable fluorescence detectors are placed on by reaction tube after fully mixing
(Twista) real-time fluorescence amplification, 37 DEG C of isothermal reaction 20min are carried out in；
The reaction system of table 1
| RPA reagents | 1 pipe tests dosage | N pipes test dosage |
| Rehydration buffer | 29.5μL | 29.5×nμL |
| RPA-F | 2.0μL | 2.0×nμL |
| RPA-R | 2.0μL | 2.0×nμL |
| RPA-P | 0.5μL | 0.5×nμL |
| Deionized water | 14.0μL | 14.0×nμL |
| DNA profiling | 2.0μL | 2.0×nμL |
| Cumulative volume | 50.0μL | 50.0×nμL |
4.2.2 blank control, negative control, positive control) are set
Negative control, blank control and positive control are respectively provided with per secondary response；
Blank control：DNA profiling is replaced with water；
Negative control：Sample, the template that extraction DNA reacts as RPA are replaced with water；
Positive control：The template that beta hemolytic streptococcus reference culture extraction DNA reacts as RPA；
4.3) result is observed
After reaction terminates, the amplification curve of observation RPA portable fluorescence detectors (Twista) generation, blank control, the moon
Property control without amplification curve, positive control has a typical S types amplification curve, judges that experiment is effective, and otherwise experiment is considered as invalid；
Step 5: result is reported
In blank control, negative control without amplification curve, on the premise of positive control has typical S types amplification curve, sample
Occur detecting beta hemolytic streptococcus in the S types amplification curve report sample of similar positive control.
Embodiment 3
The method of beta hemolytic streptococcus, comprises the following steps in a kind of quick detection textile：
Step 1: the preparation of immunomagnetic beads
0.1mL magnetic beads liquid is taken in the centrifuge tube for filling 1.0mL PBST buffer solutions, is mixed, 3000r/min centrifugation 2min,
Abandon liquid；Repeat the above steps cleaning 3 times, and adding 1.0mL PBST buffer solutions makes magnetic bead be resuspended wherein；Into above-mentioned re-suspension liquid
0.1mL beta hemolytic streptococcus immune serums are added, put shaking table, the light and slow rotational oscillation 3h of room temperature；Cleaned with 1 × washing lotions of 1.0mL
After 5 times, 0.5mL re-suspension liquids are added, are saved backup in 4 DEG C；
Step 2: RPA primers and probe design
Coding HtrA target-gene sequence is downloaded in ncbi database, sequence alignment is carried out using CLUSTAL X softwares,
One section is have found in HtrA genes only to guard in the specific sequence of A, B, C, G group of beta hemolytic streptococcus, and in this sequence
A set of specific primer and probe using RPA technologies as core technology is designed in row；
Step 3: the immunomagnetic beads enrichment of beta hemolytic streptococcus
Sterile working is weighed in 25g samples to the sterile homogenizing bag of band filter screen equipped with 100mL peptone waters, light and slow vibration
30min；Sample is filtered, supernatant is shifted in centrifuge tube, 5000r/min centrifugation 20min, abandons supernatant fluid, precipitation is resuspended in
In 1.0mL PBST buffer solutions；Whole sample suspensions are taken in the tubule equipped with 0.1mL immunomagnetic beads suspensions, the light and slow rotation of room temperature
Vibrate 30min；Liquid in pipe is carefully removed from, 1.0mL washing lotions is added and mixes, washing lotion is removed after 3min, repeated washing 5 times, is added
0.1mL re-suspension liquids make the streptococcic magnetic bead of absorption beta hemolysis be resuspended wherein；Suspension containing magnetic beads with beta hemolytic streptococcus
It can be directly used for extracting DNA of bacteria；
Step 4: RPA is identified
4.1) preparation of DNA profiling
100 μ L beta hemolytic streptococcuses suspension containing magnetic beads are taken in 0.5mL pipes, 100 DEG C of water-bath 10min, 5000r/min centrifugations
5min, supernatant is taken, -20 DEG C save backup；
4.2) RPA amplified reactions
4.2.1) RPA reaction tube numbers n (the pipe negative controls of n=1 pipes blank control+1 according to required for being set sample size
+ 1 pipe positive control+sample number)；Table is sequentially added into the 0.2mL TwistAmp Exo RPA reaction tubes containing lyophilized enzyme powder
Each composition in 1,2000r/min centrifugations 10sec, RPA portable fluorescence detectors are placed on by reaction tube after fully mixing
(Twista) real-time fluorescence amplification, 37 DEG C of isothermal reaction 20min are carried out in；
The reaction system of table 1
| RPA reagents | 1 pipe tests dosage | N pipes test dosage |
| Rehydration buffer | 29.5μL | 29.5×nμL |
| RPA-F | 2.0μL | 2.0×nμL |
| RPA-R | 2.0μL | 2.0×nμL |
| RPA-P | 0.5μL | 0.5×nμL |
| Deionized water | 14.0μL | 14.0×nμL |
| DNA profiling | 2.0μL | 2.0×nμL |
| Cumulative volume | 50.0μL | 50.0×nμL |
4.2.2 blank control, negative control, positive control) are set
Negative control, blank control and positive control are respectively provided with per secondary response；
Blank control：DNA profiling is replaced with water；
Negative control：Sample, the template that extraction DNA reacts as RPA are replaced with water；
Positive control：The template that beta hemolytic streptococcus reference culture extraction DNA reacts as RPA；
4.3) result is observed
After reaction terminates, the amplification curve of observation RPA portable fluorescence detectors (Twista) generation, blank control, the moon
Property control have amplification curve or positive control without typical S types amplification curve, it is invalid that experiment is considered as, and need to reform.
Method performance test
1) sample is prepared
1.1) sample is collected
The sample of different fiber compositions and totally 15 parts of the sample of different purposes are collected, including：New 1 part of diablement fort, silk goods 1
Part, 1 part of knitting wool clothes material, 1 part of cotton thread, 1 part of woven dacron, 1 part of blending plain cloth, 1 part of garrha, 1 part of oilcloth, 1 part of canvas, poplin cloth
1 part of cloth, 1 part of denim, 1 part of corduroy, 1 part of mull-chiffon, 1 part of silks and satins cloth, 1 part of polyamide fabric, dried after water rinses each
Part sample, which is fitted into a sterile sampler bag, does autoclaving processing.Respectively numbering 1,2,3,4 ... 15.Separately collect 5 tradition
The positive sample of method detection, including：Hospital's sheet 1,2, hospital's pillowcase, 1, quilt cover of hospital, 1 set of clothing of nurses, respectively
Numbering 16,17,18,19,20.
1.2) sample making
Take quality control standard bacterial strain beta hemolytic streptococcus CMCC32210-5a, α-hemolytic streptococcus CMCC32213, green
Purulence bacillus ATCC10145, production nitrogen pseudomonad CGMCC1.1792, salmonella london CMCC50106, staphylococcus aureus
It is ATCC29213, Listeria monocytogenes CMCC54002, Enterobacter sakazakii IQCC10403, vibrio parahemolyticus CICC21617, big
Enterobacteria CMCC44102, Escherichia coli O 157 NCTC12900, it is inoculated with 300mL nutrient broths brings back to life respectively, the meat after culture
Soup respectively takes 10mL to be mixed, and after 10 times of doubling dilutions of mixed bacteria liquid, concentration bacterium solution (10000 times of dilution) is artificial dirty in selection
1~No. 5 sample is contaminated, placement overnight, takes low concentration bacterium solution (10000000 times of dilution) 6~No. 20 samples of artificial contamination, it is overnight to put
Put.
2) specific test
With reference culture beta hemolytic streptococcus CMCC32210-5a, α-hemolytic streptococcus CMCC32213, agalasisa chain
Coccus ATCC13813, Pseudomonas aeruginosa ATCC10145, production nitrogen pseudomonad CGMCC1.1792, salmonella london
CMCC50106, staphylococcus aureus ATCC29213, Listeria monocytogenes CMCC54002, Enterobacter sakazakii IQCC10403,
Vibrio parahemolyticus CICC21617, Escherichia coli CMCC44102, Escherichia coli O 157 NCTC12900 DNA are template, are carried out
IMS- real-time fluorescence PCRs are tested, as a result such as Fig. 2.There are RPA amplification curves and raised in positive beta hemolysis streptococcus, and other
Bacterium amplification curve is smooth linear, no non-specific amplification.
3) sensitivity test
Reference culture beta hemolytic streptococcus bacterium solution is taken to extract DNA, with ultramicron ultra-violet and visible spectrophotometer
(NanoDrop) test dna concentration is 465.34ng/ μ L (such as Fig. 3).10 times are diluted to 46.534ng/ μ L, 4.6534ng/ successively
μ L, 0.46534ng/ μ L, 0.046534ng/ μ L and 0.0046534ng/ μ 5 gradients of L.Each dilution factor bacterium solution is immunized respectively
RPA amplifications are carried out after enrichment with magnetic bead.As a result such as Fig. 4.The RPA detection sensitivity amplification curves of beta hemolytic streptococcus positive strain
The time point raised as DNA profiling concentration reduces also gradually is delayed；Simultaneously RPA amplified productions fluorescence signal value with
The reduction of DNA profiling concentration and be gradually reduced.When DNA profiling concentration is 0.046534ng/ μ L, fluorescence signal remains to
In trend of raising during 20min, and when DNA profiling concentration is 0.0046534ng/ μ L, its amplification curve is relatively low flat, with negative control
It is almost similar.
4) stability test
1~No. 20 sample is detected according to rebuilding method simultaneously.Final 1~No. 20 sample all detects beta hemolysis
Streptococcus, it is consistent with expected results.Fig. 5 lists 1~No. 20 sample RPA amplification curve diagram.It is therefore seen that artificial addition no matter
It is that middle concentration level or low concentration are horizontal, rebuilding method can be expected to detect, identification rate of accuracy reached to 100%, natural pollution
Flat identification accuracy rate is allowed also to reach 100%.
It was found from above method performance test, beta hemolytic streptococcus in a kind of quick detection textile provided by the invention
Method strong interference immunity, being capable of specific detection beta hemolytic streptococcus；Detection sensitivity is high, and detection lower bound is 10CFU/
ML, the detection lower bound of other detection methods is met or exceeded；Testing result stability is strong, rate of accuracy reached to 100%.
The foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto, ability
Other modifications or equivalent substitution that domain those of ordinary skill is made to technical scheme, without departing from skill of the present invention
The spirit and scope of art scheme, it all should cover among scope of the presently claimed invention.
Claims (2)
1. a kind of method of beta hemolytic streptococcus in quick detection textile, it is characterised in that comprise the following steps：
Step 1: the preparation of immunomagnetic beads
0.1mL magnetic beads liquid is taken in the centrifuge tube for filling 1.0mL PBST buffer solutions, is mixed, 3000r/min centrifugation 2min, abandons liquid
Body；Repeat the above steps cleaning 3 times, and adding 1.0mL PBST buffer solutions makes magnetic bead be resuspended wherein；Added into above-mentioned re-suspension liquid
0.1mL beta hemolytic streptococcus immune serums, put shaking table, the light and slow rotational oscillation 3h of room temperature；Cleaned 5 times with 1 × washing lotions of 1.0mL
Afterwards, 0.5mL re-suspension liquids are added, are saved backup in 4 DEG C；
Step 2: RPA primers and probe design
Coding HtrA target-gene sequence is downloaded in ncbi database, sequence alignment is carried out using CLUSTAL X softwares,
One section is have found in HtrA genes only to guard in the specific sequence of A, B, C, G group of beta hemolytic streptococcus, and in this sequence
The a set of specific primer and probe using RPA technologies as core technology of middle design；
Step 3: the streptococcic immunomagnetic beads enrichment of beta hemolysis
Sample suspension is prepared, immunomagnetic beads enrichment, obtains the suspension containing magnetic beads with beta hemolytic streptococcus；
Step 4: RPA is identified
4.1) preparation of DNA profiling
100 μ L beta hemolytic streptococcuses suspension containing magnetic beads are taken in 0.5mL centrifuge tubes, 100 DEG C of water-bath 10min, 5000r/min centrifugations
5min, supernatant is taken, -20 DEG C save backup；
4.2) RPA amplified reactions
4.2.1) the RPA reaction tube numbers n according to required for being set sample size (manage by the pipe negative control+1 of n=1 pipes blank control+1
Positive control+sample number)；Sequentially added into the 0.2mL TwistAmp Exo RPA reaction tubes containing lyophilized enzyme powder in table 1
Each composition, 2000r/min centrifugations 10sec, RPA portable fluorescence detectors (Twista) are placed on by reaction tube after fully mixing
Middle progress real-time fluorescence amplification, 37 DEG C of isothermal reaction 20min；
The reaction system of table 1
4.2.2 blank control, negative control, positive control) are set
Negative control, blank control and positive control are respectively provided with per secondary response；
Blank control：DNA profiling is replaced with water；
Negative control：Sample, the template that extraction DNA reacts as RPA are replaced with water；
Positive control：The template that beta hemolytic streptococcus reference culture extraction DNA reacts as RPA；
4.3) result is observed
After reaction terminates, the amplification curve of observation RPA portable fluorescence detectors (Twista) generation, blank control, feminine gender are right
According to without amplification curve, positive control has typical S types amplification curve, judges that experiment is effective, and otherwise experiment is considered as invalid；
Step 5: result is reported
In blank control, negative control without amplification curve, on the premise of positive control has typical S types amplification curve, sample is without expansion
Increase in curve report sample and do not detect beta hemolytic streptococcus；There is the S types amplification curve report sample of similar positive control in sample
Beta hemolytic streptococcus is detected in product.
2. the method for beta hemolytic streptococcus in a kind of quick detection textile according to claim 1, it is characterised in that
Sample suspension is prepared in step 3, immunomagnetic beads enrichment specially sterile working weighs 25g samples and 100mL peptone waters extremely are housed
The sterile homogenizing bag of band filter screen in, light and slow vibration 30min；Sample is filtered, supernatant is shifted in centrifuge tube, 5000r/min centrifugations
20min, abandons supernatant fluid, and precipitation is resuspended in 1.0mL PBST buffer solutions；Whole sample suspensions are taken in magnetic is immunized equipped with 0.1mL
In the tubule of pearl suspension, the light and slow rotational oscillation 30min of room temperature；Liquid in pipe is carefully removed from, 1.0mL washing lotions is added and mixes, 3min
After remove washing lotion, repeated washing 5 times, adding 0.1mL re-suspension liquids makes the magnetic bead of absorption beta hemolytic streptococcus be resuspended wherein；Gained
Suspension containing magnetic beads with beta hemolytic streptococcus can be directly used for extracting DNA of bacteria.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103214A (en) * | 2018-02-10 | 2018-06-01 | 中华人民共和国浙江出入境检验检疫局 | The primed probe group and kit of RAA Fluorometric assay hemolytic streptococcus |
| CN109609604A (en) * | 2019-02-21 | 2019-04-12 | 郭会清 | Multiple IMS- fluorescence RPA detection method that is a kind of while detecting 3 kinds of zoonosis pathogens |
| CN111705105A (en) * | 2020-07-13 | 2020-09-25 | 北京海关技术中心 | Qualitative detection capability verification sample for hemolytic streptococcus in cotton swab and preparation method thereof |
| CN112940964A (en) * | 2021-01-29 | 2021-06-11 | 西南大学 | Karst trough region stony desertification soil improvement microbial agent and preparation and application thereof |
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2017
- 2017-10-27 CN CN201711027424.7A patent/CN107641643A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103214A (en) * | 2018-02-10 | 2018-06-01 | 中华人民共和国浙江出入境检验检疫局 | The primed probe group and kit of RAA Fluorometric assay hemolytic streptococcus |
| CN109609604A (en) * | 2019-02-21 | 2019-04-12 | 郭会清 | Multiple IMS- fluorescence RPA detection method that is a kind of while detecting 3 kinds of zoonosis pathogens |
| CN111705105A (en) * | 2020-07-13 | 2020-09-25 | 北京海关技术中心 | Qualitative detection capability verification sample for hemolytic streptococcus in cotton swab and preparation method thereof |
| CN112940964A (en) * | 2021-01-29 | 2021-06-11 | 西南大学 | Karst trough region stony desertification soil improvement microbial agent and preparation and application thereof |
| CN112940964B (en) * | 2021-01-29 | 2023-05-09 | 西南大学 | Karst trough area stony desertification soil improvement microbial agent, and preparation and application thereof |
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