CN110272898B - Universal microbial pathogen lysate and application thereof - Google Patents
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Abstract
The invention provides a general microbial pathogen lysate and application thereof. Specifically, the lysate comprises: 30-60mM Tris-HCl, 3-6% (w/w) nonionic surfactant, 300-600mM guanidine isothiocyanate, and 20-80mM KCl, wherein the pH of the lysate is 7.0-10.0, the percentages being based on the total weight of the lysate. The lysate of the invention is suitable for pathogen microorganisms of various sources, and has the advantages of high nucleic acid extraction speed, high extraction rate and low cost, and unexpectedly, the lysate of the invention can also improve PCR amplification efficiency.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a general microbial pathogen lysate and application thereof.
Background
Pathogens refer to disease causing microorganisms and parasites, most of which include viruses, chlamydia, mycoplasma, bacteria, fungi, and the like. Infectious diseases are non-negligible public health problems facing in common worldwide, and their incidence is also the leading one among various diseases in humans. The establishment of a rapid and effective pathogen detection method plays a vital role in the prevention, detection and treatment of infectious diseases.
The traditional microbial pathogen detection method mainly comprises a culture method, immunological detection, nucleic acid detection and the like, the culture and identification method is complex in operation, the technical level requirement on operators is high, the detection period is long, generally 5-7 days are required, and the requirements of clinical diagnosis and treatment cannot be met in time, so that the treatment of patients can be delayed. Also, in the case of epidemic outbreak of infectious diseases, laboratories cannot meet the requirements of epidemic prevention and control only by relying on traditional culture methods.
With the rapid development of nucleic acid detection technology, nucleic acid molecular diagnostics have also been pushed to unprecedented high levels. The first step in the conventional nucleic acid detection technique is to extract nucleic acid from a sample. Methods of pathogen nucleic acid extraction commonly used in detection institutions today include Trizol extraction (Life Technology), column extraction (Qiagen, roche) and magnetic bead (Chemagen, thermo, jin Maige). The yield of the nucleic acid extracted by the Trizol method is relatively low, the steps are complicated, the whole extraction process takes 1-2 hours, and the detection efficiency is directly affected. The kit of the column extraction method and the magnetic bead method improves the nucleic acid extraction efficiency to a certain extent, simplifies the nucleic acid extraction step, and improves the extraction speed, but the whole extraction process still needs 0.5-1 hour. In addition, these extraction kits are extremely expensive, resulting in high detection costs. Therefore, the clinical gene diagnosis needs a sample pretreatment reagent which is simple and quick to operate and low in cost, and is particularly important for simple and quick nucleic acid extraction especially for cases with large clinical sample quantity.
However, the existing rapid nucleic acid cleavage reagent for pathogens is single in sample type and is not suitable for diversified sample types of microbial pathogens.
Therefore, aiming at the molecular diagnosis of various clinical pathogenic microorganism infectious diseases, development of a general-purpose pathogenic microorganism (such as various bacteria, DNA/RNA viruses and the like existing in samples such as whole blood, feces, urine, throat swab and the like) lysate with high nucleic acid extraction speed and high extraction rate and low price is urgently needed.
Disclosure of Invention
The invention aims to provide a general-purpose lysate of pathogenic microorganisms, which has the advantages of high nucleic acid extraction speed, high extraction rate and low cost.
The first aspect of the invention provides a microbial pathogen lysate comprising the components:
30-60mM Tris-HCl,
3-6% (w/w) of a nonionic surfactant,
300-600mM guanidine isothiocyanate, and
20-80mM of KCl, and,
wherein the pH of the lysate is 7.0-10.0, and the percentage is based on the total weight of the lysate.
In another preferred embodiment, the lysate solvent is water.
In another preferred embodiment, 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 embodiment, the pH of the lysate is adjusted with Tris-HCl having a concentration of 0.9-1.1M and a pH of 9-10, preferably Tris-HCl having a concentration of 1M and a pH of 10.
In another preferred embodiment, the Tris-HCl concentration is 40-60mM, preferably 40-55 mM, more preferably 45-55mM.
In another preferred embodiment, the nonionic surfactant concentration is 4-6% (w/w), preferably 4-5.5% (w/w), more preferably 4.5-5.5% (w/w), based on the total weight of the lysate.
In another preferred embodiment, the nonionic surfactant concentration is 4-6% (v/v), preferably 4-5.5% (v/v), more preferably 4.5-5.5% (v/v), based on the total volume of the lysate.
In another preferred embodiment, the nonionic surfactant is selected from the group consisting of: triton X-100, tween20, or a combination thereof, preferably Triton X-100.
In another preferred embodiment, the concentration of guanidinium isothiocyanate is 400-600mM, preferably 400-550mM, most preferably 450-500mM.
In another preferred embodiment, the KCl concentration is 20-60mM, preferably 40-60mM, more preferably 40-50mM.
In another preferred embodiment, the lysate comprises:
40-60mM Tris-HCl,
4-6% (v/v) of a nonionic surfactant,
400-600mM guanidine isothiocyanate, and
20-60mM of KCl, and,
wherein the pH of the lysate is 7.0-10.0, and the percentage is based on the total volume of the lysate.
In another preferred embodiment, the lysate comprises:
45-55mM Tris-HCl,
4.5-5.5% (v/v) of a nonionic surfactant,
450-550mM guanidine isothiocyanate, and
40-60mM of KCl, and the concentration of KCl,
wherein the pH of the lysate is 7.0-10.0, and the percentage is based on the total volume of the lysate.
In another preferred embodiment, the lysate comprises:
50mM Tris-HCl,
5% (v/v) nonionic surfactant,
500mM guanidine isothiocyanate, and
40-50mM of KCl, which is used for preparing the medicine,
wherein the percentages are based on the total volume of the lysate.
In another preferred embodiment, the lysate comprises:
50mM Tris-HCl,
5% (v/v) TritonX-100,
500mM guanidine isothiocyanate, and
50mM of KCl, which was used as a reagent,
wherein the percentages are based on the total volume of the lysate.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof.
In another preferred embodiment, the microbial pathogen has DNA and/or RNA.
In a second aspect, the invention provides a microbial pathogen lysis kit comprising:
(1) A lysate according to the first aspect of the invention;
(2) At least one vessel containing the lysis solution.
In another preferred embodiment, the kit further comprises instructions.
In another preferred embodiment, each vessel contains 0.5-10mL, preferably 1-5mL, of the lysate.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof.
In another preferred embodiment, the microbial pathogen has DNA and/or RNA.
In a third aspect, the present invention provides a method for rapid lysis of a microbial pathogen for extraction of nucleic acid, the method comprising the steps of:
(1) Mixing the sample with the lysate according to the first aspect of the present invention to obtain a mixture;
(2) And incubating the mixture for a certain time, centrifuging, and collecting supernatant to obtain the nucleic acid extract.
In another preferred embodiment, the incubation time of the mixture is 1-10min, preferably 2-8min, more preferably 3-6min.
In another preferred embodiment, for liquid samples, the volume ratio of the sample to the lysate is 1:0.5-5, preferably 1:1-3, more preferably 1:1-2.
In another preferred embodiment, for solid samples, the ratio of the amount of sample to lysate (mg/. Mu.L) is 1:0.5-10, preferably 1:1-5, more preferably 1:1-3.
In another preferred embodiment, the sample is selected from the group consisting of: bacterial fluids, whole blood, serum, plasma, urine, saliva, throat swabs, stool, or combinations thereof.
In another preferred embodiment, the rotational speed of the centrifugation is 1000-5000rpm, preferably 2000-4000rpm.
In another preferred embodiment, the nucleic acid extract is used directly for PCR amplification without treatment.
In another preferred embodiment, the process comprises: removing impurities and desalting.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof.
In another preferred embodiment, the nucleic acid is DNA and/or RNA.
In another preferred example, the nucleic acid extract can be directly used for PCR amplification or fluorescent PCR detection, or can be stored at low temperature (-15 to minus 25 ℃) for a long period of time.
In a fourth aspect, the present invention provides a method for rapid lysis detection of a microbial pathogen, the method comprising the steps of:
(1) Mixing a sample with the lysate of the first aspect of the present invention to obtain a mixture;
(2) Incubating the mixture for a certain period of time, centrifuging, and collecting supernatant;
(3) And taking the supernatant as a sample for fluorescent PCR reaction, and reacting the sample with a primer and a probe specific to the sample for fluorescent PCR detection.
In another preferred embodiment, the detection is a quantitative detection and/or a qualitative detection.
In another preferred embodiment, the supernatant contains the extracted nucleic acid.
In another preferred embodiment, the supernatant is not subjected to any amplification for PCR.
In another preferred embodiment, the process comprises: removing impurities and desalting.
In another preferred embodiment, the incubation time of the mixture is 1-10min, preferably 2-8min, more preferably 3-6min.
In another preferred embodiment, for liquid samples, the volume ratio of the sample to the lysate is 1:0.5-5, preferably 1:1-3, more preferably 1:1-2.
In another preferred embodiment, for solid samples, the ratio of the amount of sample to lysate (mg/. Mu.L) is 1:0.5-10, preferably 1:1-5, more preferably 1:1-3.
In another preferred embodiment, the sample is selected from the group consisting of: bacterial fluids, whole blood, serum, plasma, urine, saliva, throat swabs, stool, or combinations thereof.
In another preferred embodiment, the rotational speed of the centrifugation is 1000-5000rpm, preferably 2000-4000rpm.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof.
In another preferred embodiment, the microbial pathogen has DNA and/or RNA.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the effect of lysates of different ratios on the nucleic acid extraction.
FIG. 2 shows the effect of KCl concentration in lysate on nucleic acid extraction.
FIG. 3 shows the results of a nucleic acid extraction sensitivity test of the lysate of one example.
Figure 4 illustrates the extraction effect of one embodiment of the lysate on a pharyngeal swab sample.
Figure 5 illustrates the extraction effect of one embodiment of the lysate on a fecal sample.
FIG. 6 shows the results of a comparison of the nucleic acid extraction effect of one example lysate with other commercially available extraction reagents.
FIG. 7 shows the effect of the lysate components and the lysis procedure on the nucleic acid extraction.
Detailed Description
Through extensive and intensive research and extensive screening and testing, the inventor develops a general microbial pathogen lysate through optimizing comprehensive performance. The general microbial pathogen lysate of the invention can realize rapid extraction of nucleic acid of pathogens from various sources clinically, the whole extraction process can be less than 10min, the extraction efficiency is greatly improved, and surprisingly, the nucleic acid extracted by using the lysate of the invention has no interference to subsequent PCR amplification, and the amplification efficiency is also enhanced. The present invention has been completed on the basis of this finding.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
As used herein, the terms "microbial pathogen lysate", "microbial pathogen universal fast lysate" and "lysate of the present invention" are used interchangeably to refer to microbial pathogen lysates of the present invention.
Microbial pathogen lysate
The invention provides a microbial pathogen lysate, which comprises the following components:
30-60mM Tris-HCl, 3-6% (w/w) nonionic surfactant, 300-600mM guanidine isothiocyanate, and 20-80mM KCl, wherein the pH of the lysate is 7.0-10.0, the percentages being based on the total weight of the lysate.
The solvent of the lysate of the present invention is water or substantially water.
In addition to the function of cracking microbial pathogens, the guanidine isothiocyanate in the lysate can inactivate DNase/RNase, so that degradation of nucleic acid is effectively prevented, the extraction rate of nucleic acid is ensured, and guanidine isocyanate can inactivate various protein residues after cracking, so that influence of the residues on PCR amplification effect is inhibited. In another preferred embodiment, the concentration of guanidinium isothiocyanate is 400-600mM, preferably 400-550mM, most preferably 450-500mM.
Tris-HCl can bring the cleavage system to a suitable pH environment.
In another preferred embodiment, the pH of the lysate is adjusted with Tris-HCl having a concentration of 0.9-1.1M and a pH of 9-10, preferably Tris-HCl having a concentration of 1M and a pH of 10.
In another preferred embodiment, the Tris-HCl concentration is 40-60mM, preferably 40-55 mM, more preferably 45-55mM.
In another preferred embodiment, 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 surfactants can disrupt the biological membrane structure, releasing the material within the membrane. The lysate of the present invention is not particularly limited to the nonionic surfactant, and nonionic surfactants commonly used in the art can be used.
In another preferred embodiment, the nonionic surfactant is selected from the group consisting of: triton X-100, tween20, or a combination thereof, preferably Triton X-100.
In another preferred embodiment, the nonionic surfactant concentration is 4-6% (w/w), preferably 4-5.5% (w/w), more preferably 4.5-5.5% (w/w), based on the total weight of the lysate.
In another preferred embodiment, the nonionic surfactant concentration is 4-6% (v/v), preferably 4-5.5% (v/v), more preferably 4.5-5.5% (v/v), based on the total volume of the lysate.
Unexpectedly, the addition of KCl further improves the nucleic acid extraction rate, and the PCR amplification efficiency, contributing to a reduction in the number of PCR amplifications, further reducing the analysis time.
In another preferred embodiment, the KCl concentration is 20-60mM, preferably 40-60mM, more preferably 40-50mM.
Typically, the lysate of the present invention is prepared by solution preparation methods commonly used in the art. In general, the components of the cracking liquid are dissolved in water according to the proportion and are uniformly mixed.
In another preferred embodiment, the lysate comprises: 50mM Tris-HCl, 5% (v/v) TritonX-100, 500mM guanidine isothiocyanate, and 50mM KCl, wherein the percentages are based on the total volume of the lysate.
Microbial pathogen lysis kit
The invention further provides a microbial pathogen lysis kit comprising:
(1) A lysate as described above;
(2) At least one vessel containing the lysis solution.
In another preferred embodiment, the kit further comprises instructions.
In another preferred embodiment, each vessel contains 0.5-10mL, preferably 1-5mL, of the lysate.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof.
In another preferred embodiment, the microbial pathogen has DNA and/or RNA.
Method for extracting nucleic acid by rapid lysis of microbial pathogen
The invention also provides a method for extracting nucleic acid by rapid lysis of microbial pathogens, which comprises the following steps:
(1) Mixing the sample with the lysate to obtain a mixture;
(2) And incubating the mixture for a certain time, centrifuging, and collecting supernatant to obtain the nucleic acid extract.
In another preferred embodiment, the incubation time of the mixture is 1-10min, preferably 2-8min, more preferably 3-6min.
In another preferred embodiment, for liquid samples, the volume ratio of the sample to the lysate is 1:0.5-5, preferably 1:1-3, more preferably 1:1-2.
In another preferred embodiment, for solid samples, the ratio of the amount of sample to lysate (mg/. Mu.L) is 1:0.5-10, preferably 1:1-5, more preferably 1:1-3.
In another preferred embodiment, the sample is selected from the group consisting of: bacterial fluids, whole blood, serum, plasma, urine, saliva, throat swabs, stool, or combinations thereof.
In another preferred embodiment, the nucleic acid extract is used directly for PCR amplification without treatment.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof.
Method for detecting microbial pathogen by rapid lysis
The invention also provides a method for detecting the rapid lysis of the microbial pathogen, which comprises the following steps:
(1) Mixing the sample with the lysate to obtain a mixture;
(2) Incubating the mixture for a certain period of time, centrifuging, and collecting supernatant;
(3) And taking the supernatant as a sample for fluorescent PCR reaction, and reacting the sample with a primer and a probe specific to the sample for fluorescent PCR detection.
In another preferred embodiment, the detection is a quantitative detection and/or a qualitative detection.
In another preferred embodiment, for liquid samples, the volume ratio of the sample to the lysate is 1:0.5-5, preferably 1:1-3, more preferably 1:1-2.
In another preferred embodiment, for solid samples, the ratio of the amount of sample to lysate (mg/. Mu.L) is 1:0.5-10, preferably 1:1-5, more preferably 1:1-3.
In another preferred embodiment, the sample is selected from the group consisting of: bacterial fluids, whole blood, serum, plasma, urine, saliva, throat swabs, stool, or combinations thereof.
In another preferred embodiment, the nucleic acid extract is used directly for PCR amplification without treatment.
In another preferred embodiment, the microbial pathogen is selected from the group consisting of: viruses, chlamydia, mycoplasma, bacteria, fungi, or combinations thereof
The main advantages of the invention include:
(1) The pathogen is cracked by the lysate, the whole nucleic acid extraction process can be less than 10min, the extraction efficiency is effectively improved, and the analysis time is saved;
(2) The lysate is suitable for different types of pathogenic microorganisms from various sample sources, and has wide applicability;
(3) The nucleic acid in the sample extracted from the lysate is low in loss, high in extraction rate, and can be directly used for PCR amplification without purification;
(4) Unexpectedly, the addition of KCl can improve the PCR amplification efficiency, thereby being beneficial to shortening the PCR amplification times and further shortening the detection time;
(5) The lysate of the invention has simple components and no expensive components, does not introduce substances influencing analysis, can improve the nucleic acid detection sensitivity, has low cost, and is suitable for detection and analysis of a large number of samples;
(6) The extraction method provided by the invention has the advantages of low price of the lysate, high extraction speed, high extraction rate, suitability for different pathogens from various sample sources, no need of expensive and complex nucleic acid extraction equipment, and only need of a pipettor and a centrifuge to finish the operation.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The materials or instruments used in the examples of the present invention are commercially available unless otherwise specified.
Example 1
Taking a culture type bacterial liquid sample of staphylococcus aureus as an example, the influence of different proportions of each component of the lysate on the nucleic acid extraction effect is tested.
(1) According to the formula of Table 1, a lysate A, a lysate B and a lysate C are respectively prepared, the solvent of the lysate is water, and the percentages are calculated by the total volume of the lysate. Wherein, tris-HCl is added with a certain amount of 1M Tris-HCl (pH=10.0) mother solution (normal temperature, 18-25 ℃), so that the concentration of Tris (Tris-hydroxymethyl aminomethane) in the lysate after constant volume is the corresponding concentration.
Table 1 lysate formulation
| Reagent species | Lysate A formulation | Formula of lysate B | Lysate C formulation |
| Tris-HCl(pH10.0) | 30mM | 50mM | 60mM |
| TritonX-100 | 3％(v/v) | 5％(v/v) | 6％(v/v) |
| Guanidine isothiocyanate | 300mM | 500mM | 600mM |
| KCl | 20mM | 50mM | 80mM |
(2) 50 mu L of staphylococcus aureus bacterial liquid is placed in a centrifuge tube (3 parts) respectively, 50 mu L of lysate A, lysate B and lysate C are added respectively, the mixture is blown and evenly mixed by a pipettor, then the mixture is incubated for 5min at room temperature, and then centrifuged for 2min at 3000rpm, and the supernatants are collected to obtain nucleic acid samples A, B, C respectively.
(3) A5. Mu.L sample of nucleic acid was taken and amplified by fluorescent PCR. Using an upstream primer SA-F having a sequence of 5'-TCATTATTCGACTAGATGTTG-3' (SEQ ID No.: 1) and a downstream primer SA-R having a sequence of 5'-CTCTTTTACTTTAGCAACCGTTG-3' (SEQ ID No.: 2) and a probe SA-P having a sequence of 5'-TTCGCTTAATTCGCTTAGGCGAT-3' (SEQ ID No.: 3), both ends of the probe being respectively bonded with a fluorogenic group FAM and a fluorescence quenching group BHQ; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), preparing a reaction system according to the instruction of a kit, wherein the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
As shown in the fluorescent quantitative PCR detection results shown in FIG. 1, the amplification effect of the formula of the lysate B is the best, which means that the nucleic acid extraction rate of the lysate B is the highest, and thus, it is found that the optimal concentration of the lysate is difficult to predict, and the increase or decrease of the concentrations of each component (such as lysate C and lysate A) leads to the decrease of the nucleic acid extraction efficiency.
Example 2
Selection of optimal concentration of KCl in microbial pathogen universal fast lysate formulation:
(1) Lysates with different KCl concentrations of 0, 20mM, 40mM, 50mM, 60mM and 80mM were prepared, and the other components and concentrations were the same as those of lysate B of example 1;
(2) Respectively placing 50 mu l of staphylococcus aureus bacterial liquid into a centrifuge tube, respectively adding 50 mu l of the lysate with different KCl concentrations (0, 20mM, 40mM, 50mM, 60mM and 80 mM), blowing and mixing uniformly by a pipettor, incubating the mixed liquid for 5min at room temperature, centrifuging at 3000rpm for 2min, and collecting the supernatant to obtain the nucleic acid sample.
(3) Mu.l of each nucleic acid sample was subjected to fluorescent PCR amplification. Using an upstream primer SA-F sequence of 5'-TCATTATTCGACTAGATGTTG-3' (SEQ ID No.: 1), a downstream primer SA-R sequence of 5'-CTCTTTTACTTTAGCAACCGTTG-3' (SEQ ID No.: 2), a probe SA-P sequence of 5'-TTCGCTTAATTCGCTTAGGCGAT-3' (SEQ ID No.: 3), and a fluorescence generating group FAM and a fluorescence quenching group BHQ respectively bonded to both ends of the probe; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), preparing a reaction system according to the instruction of a kit, wherein the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
From the results of the fluorescent quantitative PCR detection shown in FIG. 2, compared with the lysate with KCl concentration of 0, the Ct values of the lysate with KCl concentration of 20mM, 40mM, 50mM and 60mM are all significantly advanced, and the Ct value of the lysate with KCl concentration of 80mM is not significantly changed. The result shows that the addition of KCl can improve the PCR amplification efficiency, and the PCR amplification efficiency is obviously improved when the concentration of KCl is 20-60mM compared with the lysate without KCl, wherein the lysate with the concentration of KCl is 50mM, and the amplification effect is optimal.
Example 3
Preparation and application methods of the general rapid lysis kit for microbial pathogens (suitable for bacterial liquid/serum/plasma/urine detection):
(1) Preparation of a universal rapid lysis kit for microbial pathogens (50 test/kit): general type quick lysate 1 bottle (formulation as in example 1 lysate B,2.5 mL/bottle) for microbial pathogens.
(2) Sample collection: the serum/plasma/urine of the patient collected in clinic is directly adopted, or the cultured bacterial liquid is taken.
(3) The detection step comprises: mixing the sample with 50 μl of the lysate, blowing with a pipette, mixing, incubating the mixture at room temperature for 5min, centrifuging at 3000rpm for 2min, and collecting supernatant to obtain nucleic acid extract.
The nucleic acid extract can be directly used for PCR amplification or fluorescent PCR detection, or can be stored at-20deg.C for a long time.
Example 4
General rapid lysis kit for microbial pathogens and application method thereof (applicable to pharyngeal swab detection):
(1) Preparation of a universal rapid lysis kit for microbial pathogens (50 test/kit): 2 bottles of general-purpose rapid lysate of microbial pathogens (formula as in example 1 lysate B,5 mL/bottle).
(2) Sample collection: the special sampling swab is used, the back wall of the pharynx and tonsil parts on two sides are swabbed moderately hard, then the swab is rapidly placed in a sampling tube, a cotton swab rod is broken at a position close to the top end, and a tube cover is screwed and sealed to prevent dryness.
(3) The detection step comprises: placing a sample of the swab to be detected in 200 mu L of pathogenic microorganism sample lysate, fully stirring and eluting the sample on the swab, extruding the swab on the tube wall for a plurality of times, incubating for 5min at room temperature, centrifuging at 3000rpm for 2min, and collecting the supernatant to obtain the nucleic acid extract.
The nucleic acid extract can be directly used for PCR amplification or fluorescent PCR detection, or can be stored at-20deg.C for a long time.
Example 5
General rapid lysis kit for microbial pathogens and use method thereof (suitable for fecal detection):
(1) Preparation of a universal rapid lysis kit for microbial pathogens (50 test/kit): general type quick lysate 1 bottle (formulation as in example 1 lysate B,5 mL/bottle) for microbial pathogen.
(2) Sample collection: collecting 0.1-0.3g of fecal sample, immediately placing into a sterile feces collection tube, adding 1mL of sterile physiological saline, shaking, mixing, centrifuging at 8000rpm for 2min, discarding supernatant, washing the precipitate again, discarding supernatant, and re-suspending the precipitate with 1mL of sterile physiological saline.
(3) The detection step comprises: and respectively taking 100ul of sample suspension and lysate, mixing the sample suspension and the lysate uniformly by full shaking, incubating the mixture for 5min at room temperature, centrifuging the mixture for 2min at 3000rpm, and collecting the supernatant, namely the nucleic acid extract.
The nucleic acid extract can be directly used for PCR amplification or fluorescent PCR detection, or can be stored at-20deg.C for a long time.
Example 6
The sensitivity test was performed using nucleic acids extracted from bacterial solutions of staphylococcus aureus at different concentrations using the lysate of the present invention:
(1) Selecting single colony of Staphylococcus aureus cultured in plate, placing in 1mL physiological saline, mixing thoroughly, and diluting the bacterial solutions with physiological saline to 1×10 5 CFU/mL、1×10 4 CFU/mL、1×10 3 CFU/mL、1×10 2 CFU/mL、1×10CFU/mL。
(2) 50 mu L of each of the 4 dilutions of bacterial liquid is placed in a centrifuge tube, 50 mu L of lysate (the formula is as in example 1 lysate B) is added respectively, the mixture is blown and evenly mixed by a pipettor, then the mixture is incubated for 5min at room temperature, and then centrifuged for 2min at 3000rpm, and the supernatant is collected, thus obtaining the nucleic acid sample.
(3) A5. Mu.L sample of nucleic acid was subjected to fluorescent PCR amplification. Using an upstream primer SA-F sequence of 5'-TCATTATTCGACTAGATGTTG-3' (SEQ ID No.: 1), a downstream primer SA-R sequence of 5'-CTCTTTTACTTTAGCAACCGTTG-3' (SEQ ID No.: 2), a probe SA-P sequence of 5'-TTCGCTTAATTCGCTTAGGCGAT-3' (SEQ ID No.: 3), and a fluorescence generating group FAM and a fluorescence quenching group BHQ respectively bonded to both ends of the probe; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), according to the testThe kit illustrates a preparation reaction system, and the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
As can be seen from the detection results shown in FIG. 3, the detection sensitivity of the staphylococcus aureus bacterial liquid extracted from the lysate of the invention can reach 1X 10 2 CFU/mL, significantly higher than the 1X 10 achievable with the prior art 2 Up to 1X 10 4 CFU/mL, more be favorable to early detection.
Example 7
Fluorescent PCR detection of herpesvirus nucleic acids extracted from throat swabs using the lysates of the present invention:
(1) Three throat swabs of a patient infected with oral herpes virus are taken, the oral cavity wall is brushed, then the swab is rapidly placed in a sampling tube, a cotton swab rod is broken off near the top end, and a tube cover is screwed and sealed to prevent dryness.
(2) Placing the sample of the swab to be detected into 200 mu L of lysate (the formula is as in lysate B of example 1), fully stirring and eluting the sample on the swab, extruding the swab on the tube wall for a plurality of times, incubating for 5min at room temperature, centrifuging at 3000rpm for 2min, and collecting the supernatant to obtain the nucleic acid sample.
(3) A5. Mu.L sample of nucleic acid was subjected to fluorescent PCR amplification. Using an upstream primer HSV-F sequence of 5'-CGCCAGCGCTCGCACTT-3' (SEQ ID No.: 4), a downstream primer HSV-R sequence of 5'-CCGCAGGGTAAAGAAGTG-3' (SEQ ID No.: 5), a probe HSV-P sequence of 5'-ACGAACTGCGAACGCTTCG-3' (SEQ ID No.: 6), and a fluorescence generating group FAM and a fluorescence quenching group BHQ respectively bonded to both ends of the probe; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), preparing a reaction system according to the instruction of a kit, wherein the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
The test results shown in FIG. 4 show that three throat swab samples extracted from the lysate of the present invention were positive for oral herpes virus by fluorescence PCR.
Example 8
Fluorescence PCR detection of E.coli nucleic acid extracted from fecal samples using the lysate of the present invention:
(1) Collecting 0.1-0.3g of fecal sample, immediately placing into a sterile feces collection tube, adding 1mL of sterile physiological saline, shaking, mixing, centrifuging at 8000rpm for 2min, discarding supernatant, washing the precipitate again, discarding supernatant, and re-suspending the precipitate with 1mL of sterile physiological saline.
(2) Mixing the sample suspension with lysate (formula is as in example 1 lysate B) respectively, shaking thoroughly, incubating at room temperature for 5min, centrifuging at 3000rpm for 2min, and collecting supernatant to obtain nucleic acid sample.
(3) A5. Mu.L sample of nucleic acid was subjected to fluorescent PCR amplification. Using an upstream primer ECO-F sequence of 5'-TACGCCCAGTAATTCCGATTA-3' (SEQ ID No.: 7), a downstream primer ECO-R sequence of 5'-CAGAAGAAGCACCGGCTAA-3' (SEQ ID No.: 8), a probe ECO-P sequence of 5'-CGCTTGCACCCTCCRTATTACC-3' (SEQ ID No.: 9), and a fluorogenic group FAM and a fluoroquenching group BHQ respectively bonded to both ends of the probe; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), preparing a reaction system according to the instruction of a kit, wherein the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
As shown in FIG. 5, the stool sample extracted from the lysate of the present invention was detected as positive by fluorescence PCR.
Example 9
Taking a staphylococcus aureus cultured bacterial liquid sample as an example, the nucleic acid extracted from the lysate and the nucleic acid extracted from Qiagen column extraction reagent (QIAamp DNA Mini Kit, product number 51306) are used for fluorescent quantitative PCR comparison experiment results:
(1) Qiagen column extraction reagent procedure: a. taking 100 mu L of staphylococcus aureus bacterial liquid, centrifuging at 7500rpm for 5min, removing supernatant, adding 180 mu L of Buffer ATL and 20 mu L of protease K, uniformly mixing the mixture, incubating the mixture at 56 ℃ for 10min, and uniformly mixing the mixture for a plurality of times; b. adding 200 mu L Buffer AL and Vortex15s into the sample, uniformly mixing, and then incubating for 10min at 70 ℃; c. adding 200 mu L of absolute ethyl alcohol, and uniformly mixing with Vortex15 s; d. transferring the product of the previous step into a DNA binding column, and centrifuging at 8000rpm for 1min; e. 500. Mu.L Buffer AW1 was added to the binding column,centrifuging at 8000rpm for 1min; f. 500. Mu.L Buffer AW2 was added to the binding column and centrifuged at 14000rpm for 3min; g. transfer the binding column to a 1.5mL centrifuge tube and add 200. Mu.L Buffer H 2 O, standing for 1min at room temperature, centrifuging at 8000rpm for 1min, and collecting eluent to obtain nucleic acid sample.
(2) The operation steps of the lysate of the invention are as follows: 50 mu L of staphylococcus aureus bacterial liquid is placed in a centrifuge tube, 50 mu L of lysate (the formula is as in the lysate B of the example 1) is added, the mixture is blown and evenly mixed by a pipettor, then the mixture is incubated for 5min at room temperature, and then centrifuged for 2min at 3000rpm, and the supernatant is collected, thus obtaining the nucleic acid sample.
(3) A5. Mu.L sample of nucleic acid was taken and amplified by fluorescent PCR. Using an upstream primer SA-F sequence of 5'-TCATTATTCGACTAGATGTTG-3' (SEQ ID No.: 1), a downstream primer SA-R sequence of 5'-CTCTTTTACTTTAGCAACCGTTG-3' (SEQ ID No.: 2), a probe SA-P sequence of 5'-TTCGCTTAATTCGCTTAGGCGAT-3' (SEQ ID No.: 3), and a fluorescence generating group FAM and a fluorescence quenching group BHQ respectively bonded to both ends of the probe; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), preparing a reaction system according to the instruction of a kit, wherein the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
As can be seen from the results of the fluorescent quantitative PCR detection shown in FIG. 6, the Ct value of the lysate of the present invention is 3.45 times higher than that of the Qiagen column reagent, indicating that the nucleic acid yield of the lysate of the present invention is about 12 times higher than that of the Qiagen column reagent.
Example 10
The effect of the components of the lysate and the operation process of the invention on the fluorescence PCR detection effect is tested by taking the extracted and purified staphylococcus aureus DNA sample as a template:
(1) 50 mu L of the extracted and purified staphylococcus aureus DNA sample is taken, 50 mu L of lysate (the formula is as in the lysate B of the example 1) is added, the mixture is blown and evenly mixed by a pipettor, then the mixture is incubated for 5min at room temperature, and then centrifuged for 2min at 3000rpm, and the supernatant is collected, thus obtaining the nucleic acid sample 1.
(2) 50 μl of extracted and purified golden yellowStaphylococcal DNA samples were added to 50. Mu. L H 2 And O, blowing and uniformly mixing by a pipette, incubating the mixed solution for 5min at room temperature, centrifuging for 2min at 3000rpm, and collecting the supernatant, namely the nucleic acid sample 2.
(3) 50. Mu.L of the extracted and purified Staphylococcus aureus DNA sample was taken and 50. Mu. L H was added 2 And O, blowing and uniformly mixing by using a pipette to obtain the nucleic acid sample 3.
(4) mu.L of each of the nucleic acid sample 1, the nucleic acid sample 2 and the nucleic acid sample 3 was taken and then amplified by fluorescent PCR. Using an upstream primer SA-F sequence of 5'-TCATTATTCGACTAGATGTTG-3' (SEQ ID No.: 1), a downstream primer SA-R sequence of 5'-CTCTTTTACTTTAGCAACCGTTG-3' (SEQ ID No.: 2), a probe SA-P sequence of 5'-TTCGCTTAATTCGCTTAGGCGAT-3' (SEQ ID No.: 3), and a fluorescence generating group FAM and a fluorescence quenching group BHQ respectively bonded to both ends of the probe; and uses TB Green from Takara Corp TM Premix Ex Taq TM II (goods No. RR 820A), preparing a reaction system according to the instruction of a kit, wherein the amplification condition is 50 ℃ for 2min (1 cycle); 95 ℃ for 5min (1 cycle); 95℃for 10s and 55℃for 40s (40 cycles).
From the fluorescent quantitative detection results shown in FIG. 7, the Ct value of the nucleic acid sample 1 is 2.17 earlier than that of the nucleic acid sample 2, which indicates that the lysate component of the present invention can increase the PCR amplification efficiency by about 5 times; the Ct values of the nucleic acid sample 2 and the nucleic acid sample 3 are not obviously different, which indicates that the lysate and the using operation process thereof have no adverse effect on the fluorescence PCR detection effect.
In conclusion, the lysate of the invention has high sample lysis speed and high nucleic acid extraction rate, and the components of the lysate and the use process of the lysate have no adverse effect on the PCR detection effect, so that the nucleic acid extract obtained after the lysis can be directly used for PCR amplification and detection, and surprisingly, the lysate of the invention can also improve the PCR amplification efficiency and further accelerate the detection speed, and experiments prove that the lysate of the invention is effective on pathogens from various samples, has wide applicability and low cost, and is very suitable for the rapid detection of pathogens of large-scale samples.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
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Claims (4)
1. A microbial pathogen lysate, characterized in that the lysate consists of the following components:
50mM Tris-HCl,
5% v/v TritonX-100,
500mM guanidine isothiocyanate, and
50mM of KCl, which was used as a reagent,
wherein the solvent of the lysate is water and the pH is 10.0, the percentages being based on the total volume of the lysate.
2. A microbial pathogen lysis kit, the kit comprising:
(1) The lysate of claim 1;
(2) At least one vessel containing the lysis solution.
3. A method for rapid lysis of a microbial pathogen for extraction of nucleic acids, said method comprising the steps of:
(1) Mixing the sample with the lysate of claim 1 to obtain a mixture;
(2) Incubating the mixture for a period of time, centrifuging, and collecting supernatant to obtain nucleic acid extract;
wherein the sample is selected from one of: bacterial liquid, whole blood, serum, plasma, urine, saliva, throat swab and feces; and is also provided with
The microbial pathogen is selected from the group consisting of: viruses or bacteria.
4. A method for rapid lysis detection of a microbial pathogen, the method comprising the steps of:
(1) Mixing the sample with the lysate of claim 1 to obtain a mixture;
(2) Incubating the mixture for a period of time, centrifuging, and collecting a supernatant;
(3) Taking the supernatant as a sample of fluorescent PCR reaction, and reacting the sample with a primer and a probe specific to the sample for fluorescent PCR detection;
wherein the sample is selected from one of: bacterial liquid, whole blood, serum, plasma, urine, saliva, throat swab and feces; and is also provided with
The microbial pathogen is selected from the group consisting of: viruses or bacteria.
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