CN110272898A - A kind of universal microbial pathogens lysate and its application - Google Patents
A kind of universal microbial pathogens lysate and its application Download PDFInfo
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Abstract
The present invention provides a kind of universal microbial pathogens lysate and its applications.Specifically, the lysate includes: the KCl of the nonionic surfactant of Tris-HCl, 3-6% (w/w) of 30-60mM, 300-600mM guanidinium isothiocyanate and 20-80mM, wherein, the pH of lysate is 7.0-10.0, and percentage is with the lysate total weight.Lysate of the invention is suitable for the pathogen microorganism in a variety of sources, and nucleic acid extraction speed is fast, recovery rate is high, and cheap, unexpectedly, lysate of the invention can also improve PCR amplification efficiency.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of universal microbial pathogens lysate and its application.
Background technique
Pathogen refers to the microorganism and helminth that can cause disease, and wherein microorganism accounts for the overwhelming majority, including virus, clothing
Substance, mycoplasma, bacterium and fungi etc..Infectious diseases is the very important public health problem that the whole world faces jointly,
Its disease incidence also ranks first in the various diseases of the mankind.Establish quickly and effectively pathogen detection method in the pre- of infectious diseases
Anti-, detection and treatment aspect play a crucial role.
Traditional microbial pathogens detection method mainly includes cultivation, immunology detection and detection of nucleic acids etc., training
It is comparatively laborious to support identification method operation, it is relatively high to the requirement of operator's technical level, and detection cycle is long, generally requires 5-7 days
Time is not able to satisfy the demand of clinical diagnosis and treatment in timeliness, it is thus possible to treatment of the delay to patient.Equally, it is passing
Catch an illness Epidemic outbreak of disease when, laboratory, which relies only on classical culture protocols, can not meet the needs of epidemic prevention and control.
With the fast development of nucleic acid detection technique, nucleic acid molecules diagnosis has also been pushed to unprecedented high level.Often
It is exactly that nucleic acid extraction is carried out to sample with the first step of nucleic acid detection technique.The present common pathogen nucleic acid of testing agency extracts
Method include Trizol extraction method (Life Technology), column formulation (Qiagen, Roche) and paramagnetic particle method
(Chemagen, Thermo, Jin Maige).The nucleic acid yield that Trizol method is extracted is relatively low, and step is more many and diverse, entirely mentions
It takes process time-consuming 1-2 hours, directly affects detection efficiency.The kit of column formulation and paramagnetic particle method improves core to a certain extent
Sour extraction efficiency, also improves the speed of extraction at the step of simplifying nucleic acid extraction, but to still need to 0.5-1 small for entire extraction process
When.In addition, the price of these extracts kits is all prohibitively expensive, cause testing cost higher.Therefore, clinical gene diagnosis needs
Easy to operate, quick, cheap sample pre-treatments reagent, it is simple fast especially for the biggish case of clinical sample amount
The nucleic acid extraction of speed is particularly important.
And the sample type that the existing nucleic acid rapid cleavage reagent for pathogen is directed to is single, is not suitable for diversified
Microbial pathogens sample type.
Therefore, for the molecular diagnosis of clinical various types cause pathogeny imcrobe infection disease, there is an urgent need to develop one kind
Nucleic acid extraction speed is fast, recovery rate is high, cheap pathogenic microorganism universal (such as whole blood, excrement, urine, throat swab
Various types of bacteriums present in sample, DNA/RNA virus etc.) lysate.
Summary of the invention
That the object of the present invention is to provide a kind of nucleic acid extraction speed is fast, recovery rate is high, and cheap pathogenic microorganism is logical
With type lysate.
First aspect present invention provides a kind of microbial pathogens lysate, and the lysate includes component:
The Tris-HCl of 30-60mM,
The nonionic surfactant of 3-6% (w/w),
300-600mM guanidinium isothiocyanate, and
The KCl of 20-80mM,
Wherein, the pH of lysate is 7.0-10.0, and percentage is with the lysate total weight.
In another preferred example, the lysate solvent is water.
In another preferred example, the pH of the lysate is 7.0-10.0, preferably, 8.0-10.0, more preferably, 9.0-
10.0, most preferably, 10.
In another preferred example, the Tris-HCl tune that the pH value of the lysate is 0.9-1.1M through concentration and pH is 9-10
Section obtains, preferably, the Tris-HCl that concentration is 1M and pH is 10.
In another preferred example, the Tris-HCl concentration is 40-60mM, preferably, 40-55, more preferably, 45-55mM.
In another preferred example, the nonionic surfactant concentration is 4-6% (w/w), preferably, 4-5.5% (w/
W), more preferably, 4.5-5.5% (w/w), the percentage is with the lysate total weight.
In another preferred example, the nonionic surfactant concentration is 4-6% (v/v), preferably, 4-5.5% (v/
V), more preferably, 4.5-5.5% (v/v), the percentage is with the lysate total volume meter.
In another preferred example, the nonionic surfactant is selected from: TritonX-100, Tween20, or combinations thereof,
Preferably, TritonX-100.
In another preferred example, the concentration of the guanidinium isothiocyanate is 400-600mM, preferably, 400-550mM, most preferably
Ground, 450-500mM.
In another preferred example, the concentration of the KCl is 20-60mM, preferably, 40-60mM, more preferably, 40-50mM.
In another preferred example, the lysate includes:
The Tris-HCl of 40-60mM,
The nonionic surfactant of 4-6% (v/v),
400-600mM guanidinium isothiocyanate, and
The KCl of 20-60mM,
Wherein, the pH of lysate is 7.0-10.0, and the percentage is with the lysate total volume meter.
In another preferred example, the lysate includes:
The Tris-HCl of 45-55mM,
The nonionic surfactant of 4.5-5.5% (v/v),
450-550mM guanidinium isothiocyanate, and
The KCl of 40-60mM,
Wherein, the pH of lysate is 7.0-10.0, and the percentage is with the lysate total volume meter.
In another preferred example, the lysate includes:
The Tris-HCl of 50mM,
The nonionic surfactant of 5% (v/v),
500mM guanidinium isothiocyanate, and
The KCl of 40-50mM,
Wherein the percentage is with the lysate total volume meter.
In another preferred example, the lysate includes:
The Tris-HCl of 50mM,
The TritonX-100 of 5% (v/v),
500mM guanidinium isothiocyanate, and
The KCl of 50mM,
Wherein the percentage is with the lysate total volume meter.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination.
In another preferred example, the microbial pathogens have DNA and/or RNA.
Second aspect of the present invention provides a kind of microbial pathogens lytic reagent box, and the kit includes:
(1) lysate as described in the first aspect of the invention；
(2) at least one container for accommodating the lysate.
In another preferred example, the kit further includes specification.
In another preferred example, lysate described in 0.5-10mL is housed, preferably 1-5mL in each container.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination.
In another preferred example, the microbial pathogens have DNA and/or RNA.
Third aspect present invention provides a kind of method that microbial pathogens rapid cleavage extracts nucleic acid, the method packet
Include step:
(1) sample is mixed with lysate as described in the first aspect of the invention, obtains mixture；
(2) it is centrifuged after the mixture being incubated for one end time, collects supernatant, as nucleic acid extraction liquid.
In another preferred example, the mixture incubation time is 1-10min, preferably, 2-8min, more preferably, 3-
6min。
In another preferred example, for fluid sample, the volume ratio when sample is mixed with lysate is 1:0.5-5,
Preferably, 1:1-3, more preferably 1:1-2.
In another preferred example, for solid sample, the amount ratio (mg/ μ L) when the sample is mixed with lysate is 1:
0.5-10, preferably, 1:1-5, more preferably 1:1-3.
In another preferred example, the sample is selected from: bacterium solution, whole blood, serum, blood plasma, urine, saliva, throat swab, excrement
Just, or combinations thereof.
In another preferred example, the revolving speed of the centrifugation is 1000-5000rpm, preferably, 2000-4000rpm.
In another preferred example, the nucleic acid extraction liquid is directly used in PCR amplification without processing.
In another preferred example, the processing includes: removal of impurities, desalination.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination.
In another preferred example, the nucleic acid is DNA and/or RNA.
In another preferred example, the nucleic acid extraction liquid can be directly used for PCR amplification or fluorescent PCR detection, can also be placed in
(- 15~-25 DEG C) long-term preservation under low temperature.
Fourth aspect present invention provides a kind of microbial pathogens rapid cleavage detection method, and the method includes steps
It is rapid:
(1) sample is mixed with lysate described in first aspect present invention, obtains mixture；
(2) it is centrifuged after the mixture being incubated for one end time, collects supernatant；
(3) by the supernatant as the sample of Fluorescence PCR, sample and the primer and probe of sample specificity is anti-
It answers, carries out fluorescent PCR detection.
In another preferred example, described to be detected as quantitative detection and/or qualitative detection.
In another preferred example, the nucleic acid after extracting is included in the supernatant.
In another preferred example, the supernatant is used for PCR amplification without any.
In another preferred example, the processing includes: removal of impurities, desalination.
In another preferred example, the mixture incubation time is 1-10min, preferably, 2-8min, more preferably, 3-
6min。
In another preferred example, for fluid sample, the volume ratio when sample is mixed with lysate is 1:0.5-5,
Preferably, 1:1-3, more preferably 1:1-2.
In another preferred example, for solid sample, the amount ratio (mg/ μ L) when the sample is mixed with lysate is 1:
0.5-10, preferably, 1:1-5, more preferably 1:1-3.
In another preferred example, the sample is selected from: bacterium solution, whole blood, serum, blood plasma, urine, saliva, throat swab, excrement
Just, or combinations thereof.
In another preferred example, the revolving speed of the centrifugation is 1000-5000rpm, preferably, 2000-4000rpm.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination.
In another preferred example, the microbial pathogens have DNA and/or RNA.
In another preferred example, the method is non-diagnostic non-treatment.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows influence of the lysate of different ratio to nucleic acid extraction effect.
Fig. 2 shows influence of the KCl concentration to nucleic acid extraction effect in lysate.
Fig. 3 shows the nucleic acid extraction sensitivity test result of one embodiment lysate.
Fig. 4 shows the extraction effect that one embodiment lysate is directed to Pharyngeal swab samples.
Fig. 5 shows the extraction effect that one embodiment lysate is directed to fecal specimens.
Fig. 6 shows the comparison result of one embodiment lysate and other commercially available nucleic acid extraction effects for extracting reagent.
Fig. 7 shows lysate ingredient and cracks influence of the program to nucleic acid extraction effect.
Specific embodiment
The present inventor after extensive and in-depth study, by largely screening and testing, by the optimization of comprehensive performance, opens
A kind of universal microbial pathogens lysate is sent out.Universal microbial pathogens lysate of the invention, can be realized and face
The nucleic acid rapidly extracting of various source pathogen, entire extraction process are smaller than 10min on bed, greatly improve extraction effect
Rate, it is surprising that it is not only noiseless to subsequent PCR amplification using the nucleic acid that lysate of the invention extracts, but also can also
Enhance its amplification efficiency.The present invention is completed on this basis.
Term
Unless otherwise defined, whole technical terms and scientific terms used herein all have as belonging to the present invention
The normally understood identical meanings of field those of ordinary skill.
As used herein, in use, term " about " means that the value can be from enumerating in mentioning the numerical value specifically enumerated
Value changes not more than 1%.For example, as used herein, statement " about 100 " include 99 and 101 and between whole values (for example,
99.1,99.2,99.3,99.4 etc.).
As used herein, term " containing " or " including (including) " can be open, semi-enclosed and enclosed.It changes
Yan Zhi, the term also include " substantially by ... constitute " or " by ... constitute ".
As used herein, term " microbial pathogens lysate ", " the universal rapid cleavage liquid of microbial pathogens " and
" lysate of the invention " is used interchangeably, and refers to microbial pathogens lysate of the invention.
Microbial pathogens lysate
The present invention provides a kind of microbial pathogens lysates, and the lysate includes component:
Nonionic surfactant, the 300-600mM guanidinium isothiocyanate of Tris-HCl, 3-6% (w/w) of 30-60mM, and
The KCl of 20-80mM, wherein the pH of lysate is 7.0-10.0, and percentage is with the lysate total weight.
The solvent of lysate of the present invention is water or substantially water.
Guanidinium isothiocyanate is in addition to having the function of cracking microbial pathogens in lysate of the invention, additionally it is possible to make DNA
Enzyme/RNA enzyme inactivation guarantees nucleic acid extraction rate, and isocyanic acid guanidine can be after cracking to effectively prevent the degradation of nucleic acid
Various protein residue object inactivations, to inhibit influence of the residue to PCR amplification effect.In another preferred example, the different sulphur
The concentration of cyanic acid guanidine is 400-600mM, preferably, 400-550mM, most preferably, 450-500mM.
Tris-HCl can make cracking system be in suitable pH environment.
In another preferred example, the Tris-HCl tune that the pH value of the lysate is 0.9-1.1M through concentration and pH is 9-10
Section obtains, preferably, the Tris-HCl that concentration is 1M and pH is 10.
In another preferred example, the Tris-HCl concentration is 40-60mM, preferably, 40-55, more preferably, 45-55mM.
In another preferred example, the pH of the lysate is 7.0-10.0, preferably, 8.0-10.0, more preferably, 9.0-
10.0, most preferably, 10.
Nonionic surfactant enough destroys organism film structure, comes out the substance release in film.Cracking of the invention
Liquid does not specially require nonionic surfactant, and nonionic surfactant commonly used in the art can be used.
In another preferred example, the nonionic surfactant is selected from: TritonX-100, Tween20, or combinations thereof,
Preferably, TritonX-100.
In another preferred example, the nonionic surfactant concentration is 4-6% (w/w), preferably, 4-5.5% (w/
W), more preferably, 4.5-5.5% (w/w), the percentage is with the lysate total weight.
In another preferred example, the nonionic surfactant concentration is 4-6% (v/v), preferably, 4-5.5% (v/
V), more preferably, 4.5-5.5% (v/v), the percentage is with the lysate total volume meter.
Unexpectedly, the addition of KCl further improves nucleic acid extraction rate and PCR amplification efficiency, helps to shorten PCR expansion
Increase number, is further reduced analysis time.
In another preferred example, the concentration of the KCl is 20-60mM, preferably, 40-60mM, more preferably, 40-50mM.
Typically, lysate of the invention is prepared by solution allocation method commonly used in the art.In general, by of the invention
Lysate each component is dissolved in water mixing according to the ratio.
In another preferred example, the lysate include: the Tris-HCl of 50mM, 5% (v/v) TritonX-100,
The KCl of 500mM guanidinium isothiocyanate and 50mM, wherein the percentage is with the lysate total volume meter.
Microbial pathogens lytic reagent box
Invention further provides a kind of microbial pathogens lytic reagent box, the kit includes:
(1) lysate as described above；
(2) at least one container for accommodating the lysate.
In another preferred example, the kit further includes specification.
In another preferred example, lysate described in 0.5-10mL is housed, preferably 1-5mL in each container.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination.
In another preferred example, the microbial pathogens have DNA and/or RNA.
The method of microbial pathogens rapid cleavage extraction nucleic acid
The present invention also provides a kind of methods that microbial pathogens rapid cleavage extracts nucleic acid, and the method includes steps
It is rapid:
(1) sample is mixed with lysate as described above, obtains mixture；
(2) it is centrifuged after the mixture being incubated for one end time, collects supernatant, as nucleic acid extraction liquid.
In another preferred example, the mixture incubation time is 1-10min, preferably, 2-8min, more preferably, 3-
6min。
In another preferred example, for fluid sample, the volume ratio when sample is mixed with lysate is 1:0.5-5,
Preferably, 1:1-3, more preferably 1:1-2.
In another preferred example, for solid sample, the amount ratio (mg/ μ L) when the sample is mixed with lysate is 1:
0.5-10, preferably, 1:1-5, more preferably 1:1-3.
In another preferred example, the sample is selected from: bacterium solution, whole blood, serum, blood plasma, urine, saliva, throat swab, excrement
Just, or combinations thereof.
In another preferred example, the nucleic acid extraction liquid is directly used in PCR amplification without processing.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination.
Microbial pathogens rapid cleavage detection method
The present invention also provides a kind of microbial pathogens rapid cleavage detection method, the method includes the steps:
(1) sample is mixed with lysate as described above, obtains mixture；
(2) it is centrifuged after the mixture being incubated for one end time, collects supernatant；
(3) by the supernatant as the sample of Fluorescence PCR, sample and the primer and probe of sample specificity is anti-
It answers, carries out fluorescent PCR detection.
In another preferred example, described to be detected as quantitative detection and/or qualitative detection.
In another preferred example, for fluid sample, the volume ratio when sample is mixed with lysate is 1:0.5-5,
Preferably, 1:1-3, more preferably 1:1-2.
In another preferred example, for solid sample, the amount ratio (mg/ μ L) when the sample is mixed with lysate is 1:
0.5-10, preferably, 1:1-5, more preferably 1:1-3.
In another preferred example, the sample is selected from: bacterium solution, whole blood, serum, blood plasma, urine, saliva, throat swab, excrement
Just, or combinations thereof.
In another preferred example, the nucleic acid extraction liquid is directly used in PCR amplification without processing.
In another preferred example, the microbial pathogens are selected from: virus, Chlamydia, mycoplasma, bacterium, fungi or its
Combination
Main advantages of the present invention include:
(1) lysate of the invention cracking pathogen speed is fast, and entire nucleic acid extraction process is smaller than 10min, effectively mentions
High extracting efficiency saves analysis time；
(2) lysate of the invention is suitable for different types of pathogenic microorganism of various sample sources, and applicability is wide；
(3) lysate of the invention extracts the nucleic acid in sample and loses small, recovery rate height, and can directly use without purifying
In PCR amplification；
(4) unexpectedly, the addition of KCl can be improved PCR amplification efficiency, helps to shorten PCR amplification number, further contract
Short detection time；
(5) lysate component of the invention is simple and without expensive component, does not introduce the substance of impact analysis, core can be improved
Sour detection sensitivity, and it is low in cost, it is suitble to the detection and analysis of batch samples；
(6) extracting method lysate of the invention is cheap, and extraction rate is fast, and recovery rate is high, various samples is suitble to
The different pathogens in source, and it is not necessarily to costly, complicated nucleic acid extraction instrument, it is only necessary to operation can be completed in pipettor and centrifuge.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are weight percent and weight
Number.Raw materials used or instrument in the embodiment of the present invention, if not it illustrates, it is commercially available.
Embodiment 1
By taking the cultivation type bacteria liquid sample of staphylococcus aureus as an example, the different ratio of each ingredient of lysate of the present invention is tested
Influence to nucleic acid extraction effect.
(1) it is formulated according to table 1, prepares lysate A, lysate B, lysate C respectively, lysate solvent is water, percentage
With lysate total volume meter.Wherein Tris-HCl is that a certain amount of 1M tris-HCl (pH=10.0) mother liquor (room temperature, 18- is added
25 DEG C), the concentration respective concentration of Tris (trishydroxymethylaminomethane) in the lysate after making constant volume.
Table 1 cracks formula of liquid
| Reagent type | Lysate A formula | Lysate B formula | Lysate C formula |
| Tris-HCl(pH10.0) | 30mM | 50mM | 60mM |
| TritonX-100 | 3% (v/v) | 5% (v/v) | 6% (v/v) |
| Guanidinium isothiocyanate | 300mM | 500mM | 600mM |
| KCl | 20mM | 50mM | 80mM |
(2) 50 μ L staphylococcus aureus bacterium solutions is respectively taken to be placed in centrifuge tube (totally 3 parts), be separately added into 50 μ L lysate A,
Lysate B, lysate C are blown and beaten with pipettor and are mixed, mixed liquor be then incubated for 5min at room temperature, then 3000rpm
Lower centrifugation 2min collects supernatant, respectively nucleic acid samples A, B, C.
(3) 5 μ L nucleic acid samples is taken to carry out fluorescent PCR amplification respectively.Using upstream primer SA-F sequence be 5 '-
TCATTATTCGACTAGATGTTG-3 ' (SEQ ID No.:1) downstream primer SA-R sequence be 5 '-
CTCTTTTACTTTAGCAACCGTTG-3 ' (SEQ ID No.:2), probe SA-P sequence be 5 '-
Group FAM occurs respectively in connection with there is fluorescence for TTCGCTTAATTCGCTTAGGCGAT-3 ' (SEQ ID No.:3), the both ends of probe
With fluorescent quenching group BHQ；And use the TB Green of Takara companyTM Premix Ex TaqTMII (article No. RR820A),
Illustrate to prepare reaction system according to kit, amplification condition is 50 DEG C of 2min (1cycle)；95℃5min(1cycle)；95℃
10s, 55 DEG C of 40s (40cycles).
From fluorescence quantitative PCR detection shown in FIG. 1 the results show that the expanding effect of lysate B formula is best, illustrate to crack
The nucleic acid extraction rate highest of liquid B, it can be seen that, the optium concentration of lysate is difficult to it is anticipated that increasing or decreasing each component concentration (such as
Lysate C, lysate A) it can all lead to the reduction of nucleic acid extraction efficiency.
Embodiment 2
The selection of the optium concentration of KCl in the universal rapid cleavage formula of liquid of microbial pathogens:
(1) prepare the 0, lysate of 20mM, 40mM, 50mM, 60mM, 80mM difference KCl concentration, other components and concentration with
1 lysate B of embodiment is identical；
(2) it takes 50 μ l staphylococcus aureus bacterium solutions to be placed in centrifuge tube respectively, and is separately added into 50 μ l difference KCl concentration
The above-mentioned lysate of (0,20mM, 40mM, 50mM, 60mM, 80mM) is blown and beaten with pipettor and is mixed, then by mixed liquor in room temperature
Under the conditions of be incubated for 5min, be then centrifuged 2min under 3000rpm, collect supernatant to get nucleic acid samples are arrived.
(3) 5 μ l nucleic acid samples is taken to carry out fluorescent PCR amplification respectively.Using upstream primer SA-F sequence be 5 '-
TCATTATTCGACTAGATGTTG-3 ' (SEQ ID No.:1), downstream primer SA-R sequence be 5 '-
CTCTTTTACTTTAGCAACCGTTG-3 ' (SEQ ID No.:2), probe SA-P sequence be 5 '-
Group FAM occurs respectively in connection with there is fluorescence for TTCGCTTAATTCGCTTAGGCGAT-3 ' (SEQ ID No.:3), the both ends of probe
With fluorescent quenching group BHQ；And use the TB Green of Takara companyTMPremix Ex TaqTMII (article No. RR820A), is pressed
Illustrate to prepare reaction system according to kit, amplification condition is 50 DEG C of 2min (1cycle)；95℃5min(1cycle)；95 DEG C of 10s,
55℃40s(40cycles)。
From fluorescence quantitative PCR detection shown in Fig. 2 the results show that KCl concentration is compared with KCl concentration is 0 lysate
The lysate Ct value of 20mM, 40mM, 50mM, 60mM have obviously in advance, and the lysate Ct value that KCl concentration is 80mM becomes without obvious
Change.The result shows that the addition of KCl can be improved PCR amplification efficiency, when KCl concentration is 20-60mM, PCR amplification efficiency is not relatively for this
The lysate of addition KCl is significantly improved, and wherein KCl concentration is that the lysate expanding effect of 50mM is best.
Embodiment 3
The universal rapid cleavage kit preparation of microbial pathogens and its application method (are suitable for bacterium solution/serum/blood
Slurry/urine detection):
(1) prepare the universal rapid cleavage kit of microbial pathogens (50 tests/box): microbial pathogens are universal
1 bottle of rapid cleavage liquid (formula such as embodiment 1 lysate B, 2.5mL/ bottle).
(2) collection of specimens: patients serum/blood plasma/urine of clinical collection is directlyed adopt, or takes the bacterium solution of culture.
(3) detecting step: respectively taking 50 μ L to mix in sample with lysate, is blown and beaten and is mixed with pipettor, then will mixed
It closes liquid and is incubated for 5min at room temperature, 2min is then centrifuged under 3000rpm, collect supernatant, as nucleic acid extractive.
The nucleic acid extractive can be directly used for PCR amplification or fluorescent PCR detection, can also be placed in -20 DEG C of long-term preservations.
Embodiment 4
The universal rapid cleavage kit of microbial pathogens and its application method (being suitable for throat swab to detect):
(1) prepare the universal rapid cleavage kit of microbial pathogens (50 tests/box): microbial pathogens are universal
2 bottles of rapid cleavage liquid (formula such as embodiment 1 lysate B, 5mL/ bottle).
(2) collection of specimens: using dedicated sampling swab, and appropriateness firmly swabs pharynx rear wall and two sides almond body region, so
Swab is put in sampling pipe rapidly afterwards, is fractureed cotton swab bar by proximal portion, is screwed pipe lid and seal, with anti-drying.
(3) detecting step: swab samples to be checked are placed in the pathogenic microorganism sample dissociation liquid of 200 μ L, are sufficiently stirred and are washed
Sample on de- swab abandons swab in tube wall extruding afterwards for several times, is incubated for 5min at room temperature, then 3000rpm is centrifuged
2min collects supernatant, as nucleic acid extractive.
The nucleic acid extractive can be directly used for PCR amplification or fluorescent PCR detection, can also be placed in -20 DEG C of long-term preservations.
Embodiment 5
The universal rapid cleavage kit of microbial pathogens and its application method (being suitable for excrement to detect):
(1) prepare the universal rapid cleavage kit of microbial pathogens (50 tests/box): microbial pathogens are universal
1 bottle of rapid cleavage liquid (formula such as embodiment 1 lysate B, 5mL/ bottle).
(2) collection of specimens: acquisition 0.1-0.3g fecal specimens, be immediately placed in after acquisition in sterile urine collector, be added 1mL without
Bacterium physiological saline, oscillation mix, and 8000rpm is centrifuged 2min, abandon supernatant, repeat above step and wash precipitating again, after abandoning supernatant
It is resuspended and is precipitated with 1mL sterile saline.
(3) detecting step: taking 100ul to mix sample suspension respectively and sufficiently oscillation mixed with lysate, incubation at room temperature
5min, then 3000rpm is centrifuged 2min, collects supernatant, as nucleic acid extractive.
The nucleic acid extractive can be directly used for PCR amplification or fluorescent PCR detection, can also be placed in -20 DEG C of long-term preservations.
Embodiment 6
The nucleic acid progress extracted using lysate of the invention from the bacterium solution of the staphylococcus aureus of various concentration is sensitive
Degree test:
(1) the staphylococcus aureus single colonie of picking plate culture, is placed in 1mL physiological saline and mixes well, so
Afterwards by bacterium solution respectively with normal saline dilution to 1 × 105CFU/mL、1×104CFU/mL、1×103CFU/mL、1×102CFU/
mL、1×10CFU/mL。
(2) bacterium solution of 4 dilutions more than respectively takes 50 μ L to be placed in centrifuge tube, and being separately added into 50 μ L lysates, (formula is such as
1 lysate B of embodiment), it is blown and beaten and is mixed with pipettor, mixed liquor is then incubated for 5min at room temperature, then 3000rpm
Lower centrifugation 2min collects supernatant, as nucleic acid samples.
(3) 5 μ L nucleic acid samples is taken to carry out fluorescent PCR amplification.Using upstream primer SA-F sequence be 5 '-
TCATTATTCGACTAGATGTTG-3 ' (SEQ ID No.:1), downstream primer SA-R sequence be 5 '-
CTCTTTTACTTTAGCAACCGTTG-3 ' (SEQ ID No.:2), probe SA-P sequence be 5 '-
Group FAM occurs respectively in connection with there is fluorescence for TTCGCTTAATTCGCTTAGGCGAT-3 ' (SEQ ID No.:3), the both ends of probe
With fluorescent quenching group BHQ；And use the TB Green of Takara companyTM Premix Ex TaqTMII (article No. RR820A),
Illustrate to prepare reaction system according to kit, amplification condition is 50 DEG C of 2min (1cycle)；95℃5min(1cycle)；95℃
10s, 55 DEG C of 40s (40cycles).
For testing result as shown in Figure 3 it is found that lysate of the present invention extracts staphylococcus aureus bacterium solution, detection is sensitive
Degree is up to 1 × 102CFU/mL is significantly higher than the prior art generally reaches 1 × 102To 1 × 104CFU/mL, be more advantageous to and
Early detection.
Embodiment 7
Fluorescent PCR detection is carried out to the herpesviral nucleic acid extracted in throat swab using lysate of the invention:
(1) throat swab of three HSV of Oral Swab in Healthy People infected patients is taken, then swab is put sampling rapidly by grooming oral cavity wall
Guan Zhong fractures cotton swab bar by proximal portion, screws pipe lid and seal, with anti-drying.
(2) swab samples to be checked are placed in the lysate (formula such as 1 lysate B of embodiment) of 200 μ L, are sufficiently stirred and wash
Sample on de- swab abandons swab in tube wall extruding afterwards for several times, is incubated for 5min at room temperature, then 3000rpm is centrifuged
2min collects supernatant, as nucleic acid samples.
(3) 5 μ L nucleic acid samples is taken to carry out fluorescent PCR amplification.Using upstream primer HSV-F sequence be 5 '-
CGCCAGCGCTCGCACTT-3 ' (SEQ ID No.:4), downstream primer HSV-R sequence are 5 '-CCGCAGGGTAAAGAAGTG-
3 ' (SEQ ID No.:5), probe HSV-P sequence are 5 '-ACGAACTGCGAACGCTTCG-3 ' (SEQ ID No.:6), probe
Both ends respectively in connection with have fluorescence occur group FAM and fluorescent quenching group BHQ；And use the TB Green of Takara companyTM
Premix Ex TaqTMII (article No. RR820A) illustrates preparation reaction system according to kit, and amplification condition is 50 DEG C of 2min
(1cycle)；95℃5min(1cycle)；95 DEG C of 10s, 55 DEG C of 40s (40cycles).
Testing result shown in Fig. 4 shows that three parts of Pharyngeal swab samples that lysate of the present invention extracts carry out fluorescent PCR detection
It is the HSV of Oral Swab in Healthy People positive.
Embodiment 8
Fluorescent PCR detection is carried out to the Escherichia coli nucleic acid extracted in fecal specimens using lysate of the invention:
(1) 0.1-0.3g fecal specimens are acquired, are immediately placed in after acquisition in sterile urine collector, 1mL sterile physiological salt is added
Water, oscillation mixing, 8000rpm are centrifuged 2min, abandon supernatant, repeat above step and wash precipitating again, use 1mL sterile after abandoning supernatant
Precipitating is resuspended in physiological saline.
(2) by sample suspension, (formula is as 1 lysate B of embodiment) takes 100 μ L to mix respectively and sufficiently vibrates with lysate
It mixes, is incubated at room temperature 5min, then 3000rpm is centrifuged 2min, collects supernatant, as nucleic acid samples.
(3) 5 μ L nucleic acid samples is taken to carry out fluorescent PCR amplification.Using upstream primer ECO-F sequence be 5 '-
TACGCCCAGTAATTCCGATTA-3 ' (SEQ ID No.:7), downstream primer ECO-R sequence be 5 '-
CAGAAGAAGCACCGGCTAA-3 ' (SEQ ID No.:8), probe ECO-P sequence be 5 '-
CGCTTGCACCCTCCRTATTACC-3 ' (SEQ ID No.:9), the both ends of probe respectively in connection with have fluorescence occur group FAM and
Fluorescent quenching group BHQ；And use the TB Green of Takara companyTM Premix Ex TaqTMII (article No. RR820A), is pressed
Illustrate to prepare reaction system according to kit, amplification condition is 50 DEG C of 2min (1cycle)；95℃5min(1cycle)；95 DEG C of 10s,
55℃40s(40cycles)。
Testing result as shown in Figure 5 is it is found that the fecal specimens progress fluorescent PCR that lysate of the present invention extracts is detected as greatly
Enterobacteria is positive.
Embodiment 9
By taking the cultivation type bacteria liquid sample of staphylococcus aureus as an example, nucleic acid and Qiagen that lysate of the invention extracts
The nucleic acid that column proposes reagent (QIAamp DNA Mini Kit, article No. 51306) extraction is used for the comparative experiments knot of quantitative fluorescent PCR
Fruit:
(1) Qiagen column proposes reagent operation step: a. takes 100 μ L staphylococcus aureus bacterium solutions, 7500rpm centrifugation
5min removes supernatant, and 180 μ L Buffer ATL, 20 μ L Proteinase K, vortex 15s are added and mix, then incubate for 56 DEG C
10min is educated, centre mixes for several times；B. 200 μ L Buffer AL are added in step sample upwards, Vortex15s is mixed, then 70
DEG C be incubated for 10min；C. 200 μ L dehydrated alcohols are added, Vortex 15s is mixed；D. upper step product is transferred in DNA column,
8000rpm is centrifuged 1min；E. 500 μ L Buffer AW1,8000rpm are added into column and are centrifuged 1min；F. into column
500 μ L Buffer AW2,14000rpm are added and are centrifuged 3min；G. column is transferred in 1.5mL centrifuge tube, 200 μ L is added
Buffer H2O, after being placed at room temperature for 1min, 8000rpm is centrifuged 1min elution, the eluent of collection, as nucleic acid samples.
(2) lysate operating procedure of the present invention: taking 50 μ L staphylococcus aureus bacterium solutions to be placed in centrifuge tube, and is added 50
(formula such as 1 lysate B of embodiment), is blown and beaten with pipettor and is mixed, be then incubated for mixed liquor at room temperature μ L lysate
Then 5min is centrifuged 2min under 3000rpm, collect supernatant, as nucleic acid samples.
(3) 5 μ L nucleic acid samples is taken to carry out fluorescent PCR amplification respectively.Using upstream primer SA-F sequence be 5 '-
TCATTATTCGACTAGATGTTG-3 ' (SEQ ID No.:1), downstream primer SA-R sequence be 5 '-
CTCTTTTACTTTAGCAACCGTTG-3 ' (SEQ ID No.:2), probe SA-P sequence be 5 '-
Group FAM occurs respectively in connection with there is fluorescence for TTCGCTTAATTCGCTTAGGCGAT-3 ' (SEQ ID No.:3), the both ends of probe
With fluorescent quenching group BHQ；And use the TB Green of Takara companyTM Premix Ex TaqTMII (article No. RR820A),
Illustrate to prepare reaction system according to kit, amplification condition is 50 DEG C of 2min (1cycle)；95℃5min(1cycle)；95℃
10s, 55 DEG C of 40s (40cycles).
From fluorescence quantitative PCR detection result shown in fig. 6 it is found that the Ct value ratio Qiagen column of lysate of the present invention mentions reagent
Ct value shift to an earlier date 3.45, illustrate that the nucleic acid yield ratio Qiagen column of lysate of the present invention mentions that reagent is high, and about Qiagen column proposes examination
12 times of agent.
Embodiment 10
Using the L-form staphylococcus aureus sample for extracting and purifying as template, lysate ingredient and behaviour of the invention are tested
Make influence of the process to fluorescent PCR detection effect:
(1) the L-form staphylococcus aureus sample for taking 50 μ L to extract and purifying, and 50 μ L lysates are added and (are formulated strictly according to the facts
Apply 1 lysate B of example), it is blown and beaten and is mixed with pipettor, mixed liquor is then incubated for 5min at room temperature, then under 3000rpm
It is centrifuged 2min, collects supernatant, as nucleic acid samples 1.
(2) the L-form staphylococcus aureus sample for taking 50 μ L to extract and purifying, and 50 μ L H are added2O, pipettor piping and druming
It mixes, mixed liquor is then incubated for 5min at room temperature, 2min is then centrifuged under 3000rpm, collect supernatant, as nucleic acid
Sample 2.
(3) the L-form staphylococcus aureus sample for taking 50 μ L to extract and purifying, and 50 μ L H are added2O, pipettor piping and druming
It mixes, as nucleic acid samples 3.
(4) 5 μ L nucleic acid samples 1, nucleic acid samples 2, nucleic acid samples 3 are taken respectively, then carry out fluorescent PCR amplification respectively.Make
It is 5 '-TCATTATTCGACTAGATGTTG-3 ' (SEQ ID No.:1), downstream primer SA-R sequence with upstream primer SA-F sequence
Be classified as 5 '-CTCTTTTACTTTAGCAACCGTTG-3 ' (SEQ ID No.:2), probe SA-P sequence be 5 '-
Group FAM occurs respectively in connection with there is fluorescence for TTCGCTTAATTCGCTTAGGCGAT-3 ' (SEQ ID No.:3), the both ends of probe
With fluorescent quenching group BHQ；And use the TB Green of Takara companyTM Premix Ex TaqTMII (article No. RR820A),
Illustrate to prepare reaction system according to kit, amplification condition is 50 DEG C of 2min (1cycle)；95℃5min(1cycle)；95℃
10s, 55 DEG C of 40s (40cycles).
Fluorogenic quantitative detection shown in Fig. 7 the results show that nucleic acid samples 1 Ct value than nucleic acid samples 2 shift to an earlier date 2.17, say
Bright lysate ingredient of the present invention can make PCR amplification efficiency improve about 5 times；The Ct value of nucleic acid samples 2 and nucleic acid samples 3 is without obvious
Difference is illustrated this lysate and its is had no adverse effect using operating process to fluorescent PCR detection effect.
In conclusion lysate of the invention is fast to sample dissociation speed, nucleic acid extraction rate is high, and lysate of the present invention
Ingredient and its use process have no adverse effect to PCR detection effect, and the nucleic acid extraction liquid obtained after cracking is directly used
In PCR amplification and detection, unexpectedly, lysate of the invention can also improve PCR amplification efficiency, further speed up detection speed
Degree, and it is demonstrated experimentally that lysate of the invention is all effective to the pathogen of various sample sources, applicability is wide, and it is low in cost, it is non-
Often it is suitble to the pathogen of extensive sample quickly to detect.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
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Claims (10)
1. a kind of microbial pathogens lysate, which is characterized in that the lysate includes component:
The Tris-HCl of 30-60mM,
The nonionic surfactant of 3-6% (w/w),
300-600mM guanidinium isothiocyanate, and
The KCl of 20-80mM,
Wherein, the pH of lysate is 7.0-10.0, and percentage is with the lysate total weight.
2. lysate as described in claim 1, which is characterized in that the lysate solvent is water.
3. lysate as described in claim 1, which is characterized in that the concentration of the KCl is 20-60mM, preferably, 40-
60mM, more preferably, 40-50mM.
4. lysate as described in claim 1, which is characterized in that the lysate includes:
The Tris-HCl of 50mM,
The nonionic surfactant of 5% (v/v),
500mM guanidinium isothiocyanate, and
The KCl of 40-50mM,
Wherein the percentage is with the lysate total volume meter.
5. lysate as described in claim 1, which is characterized in that the lysate includes:
The Tris-HCl of 50mM,
The TritonX-100 of 5% (v/v),
500mM guanidinium isothiocyanate, and
The KCl of 50mM,
Wherein the percentage is with the lysate total volume meter.
6. a kind of microbial pathogens lytic reagent box, which is characterized in that the kit includes:
(1) lysate as described in claim 1；
(2) at least one container for accommodating the lysate.
7. a kind of method that microbial pathogens rapid cleavage extracts nucleic acid, which is characterized in that the method includes the steps:
(1) sample is mixed with lysate as described in claim 1, obtains mixture；
(2) it is centrifuged after the mixture being incubated for one end time, collects supernatant, as nucleic acid extraction liquid.
8. the method for claim 7, which is characterized in that the sample is selected from: bacterium solution, whole blood, serum, blood plasma, urine,
Saliva, throat swab, excrement, or combinations thereof.
9. the method for claim 7, which is characterized in that the microbial pathogens are selected from: virus, Chlamydia, Zhi Yuan
Body, bacterium, fungi, or combinations thereof.
10. a kind of microbial pathogens rapid cleavage detection method, which is characterized in that the method includes the steps:
(1) sample is mixed with lysate as described in claim 1, obtains mixture；
(2) it is centrifuged after the mixture being incubated for one end time, collects supernatant；
(3) by the supernatant as the sample of Fluorescence PCR, sample is reacted with the primer and probe of sample specificity,
Carry out fluorescent PCR detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164207A (en) * | 2022-02-11 | 2022-03-11 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
| CN114276932A (en) * | 2022-02-12 | 2022-04-05 | 合肥巅峰生物科技有限公司 | Microbial cell lysate |
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|---|---|---|---|---|
| US20110059455A1 (en) * | 2009-09-03 | 2011-03-10 | Becton, Dickinson And Company | Methods and compositions for direct chemical lysis |
| US20120231446A1 (en) * | 2009-11-19 | 2012-09-13 | Qiagen Gmbh | Method for selectively enriching and isolating microbial and optionally additional viral nucleic acids |
| WO2016024263A1 (en) * | 2014-08-14 | 2016-02-18 | Molecular Detection Israel Ltd. | Methods for isolating microbial dna from a blood sample |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110059455A1 (en) * | 2009-09-03 | 2011-03-10 | Becton, Dickinson And Company | Methods and compositions for direct chemical lysis |
| CN102575220A (en) * | 2009-09-03 | 2012-07-11 | 贝克顿·迪金森公司 | Methods and compositions for direct chemical lysis |
| US20120231446A1 (en) * | 2009-11-19 | 2012-09-13 | Qiagen Gmbh | Method for selectively enriching and isolating microbial and optionally additional viral nucleic acids |
| WO2016024263A1 (en) * | 2014-08-14 | 2016-02-18 | Molecular Detection Israel Ltd. | Methods for isolating microbial dna from a blood sample |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114164207A (en) * | 2022-02-11 | 2022-03-11 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
| CN114164207B (en) * | 2022-02-11 | 2022-05-17 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
| CN114276932A (en) * | 2022-02-12 | 2022-04-05 | 合肥巅峰生物科技有限公司 | Microbial cell lysate |
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