CN112626183B - Method for detecting genome damage after ultraviolet irradiation - Google Patents
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Abstract
A method for detecting genome damage after ultraviolet irradiation is used for quantitatively detecting microbial genome damage by a dual-channel TaqMan fluorescent probe and comprises the following preparation steps: culturing with corresponding culture medium to obtain required microbial culture solution; quantitatively obtaining a supernatant obtained by splitting the microbial culture solution to perform fluorescence quantitative PCR amplification as a control group and an experimental group template respectively, wherein the experimental group culture solution is irradiated under certain ultraviolet intensity; collecting the fluorescence signal CT values of the first control group, the second control group and each subgroup of the experimental group; further obtaining the standard deviation s and the detection limit L of the delta CTCK values of all the subgroups of the second control group, the CT difference value (delta CTuv) of all the subgroups of the experimental group and the average value (delta CTCK-) of the CT difference value of all the subgroups of the first control group; Δ Δ CT = Δ CTuv- Δ CTCK-as a criterion for group injury. The method for quantitatively detecting the microbial genome damage by using the fluorescent probe has stronger specificity and higher sensitivity, and can be used for quickly detecting the identification of the microbial genome damage after ultraviolet irradiation.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a method for detecting genome damage after ultraviolet irradiation.
Background
Ultraviolet (UV) irradiation can enable adjacent pyrimidine bases in DNA to form pyrimidine dimers which are connected in a covalent bond mode, mainly comprises Cyclobutane Pyrimidine Dimer (CPD), pyrimidine (6-4) pyrimidone photoproduct ((6-4) PP) and Dewar valence bond isomers, influences the double helix structure of the DNA, enables the replication and transcription functions of the DNA to be blocked, and finally plays a role in inactivating pests in the environment. Therefore, the method for rapidly detecting the damage degree of the biological genome after ultraviolet irradiation is provided, and the method has very important significance for accurately and rapidly detecting the DNA damage degree of the organism in the environment after ultraviolet irradiation.
PCR is also known as polymerase chain reaction, and uses molecular techniques to simulate the replication of nucleic acids in vivo. The fluorescent quantitative PCR is to mark and track the PCR product through fluorescent dye or fluorescent marked specific probe, monitor the reaction process in real time and on-line, detect the change of the amount of each cycle of amplified product in the PCR amplification reaction in real time through the change of fluorescent signal, quantitatively analyze the initial template through the analysis of CT value and standard curve, and quantify the initial template in the exponential phase of amplification.
The concept in the fluorescent quantitative PCR technology-CT value, C stands for Cycle, T stands for threshold, and the meaning of CT value is: the number of cycles that the fluorescence signal in each reaction tube has undergone to reach a set threshold.
In the fluorescent quantitative PCR experiment, a certain corresponding relation exists between the quantity of the initial template DNA and the CT value of the experiment. If the microorganism is subjected to ultraviolet radiation, damage to the genomic DNA will result in a change in the amount of DNA template at the beginning of the experiment, giving a different CT value. To determine whether a sample has been subjected to UV radiation, two different DNA fragments are amplified using two different fluorescent probes in a reaction system: one is a short fragment (60-300 bp), and the damage probability of the short fragment is smaller, so that the CT value change is smaller with the increase of the ultraviolet radiation dose, and the short fragment can be used as a reference group; the other is a long fragment (1000-.
At present, the DNA damage detection technology represented by comet assay is mainly used for detecting DNA damage of eukaryotic cells, and has complex operation and long detection time. The methods based on the detection of pyrimidine dimers are enzymatic methods for identifying the site of damage, and a series of antibody-based immunization methods derived using specific antibodies that recognize pyrimidine dimers, such as ELISA, immunoblotting, etc., as well as mass-spectrometric coupling based on hydrolyzed DNA. Wherein, the enzymolysis method has complex operation and more processes, and needs running glue to combine radioactive or fluorescent labels. The antibody method has the advantages of multiple steps, long time and higher requirement on the skills of detection personnel. The chromatographic mass spectrometry coupling method has complex sample preparation and expensive instruments. These methods cannot meet the requirement of rapid detection of DNA damage in a short time, and are not time-saving and convenient for real-time detection. Therefore, it is necessary and desirable to rapidly detect the damage of the genomic DNA of the microorganism after uv irradiation by using the fluorescent quantitative PCR technique, to shorten the detection time, and to improve the sensitivity.
Disclosure of Invention
In order to solve at least one problem mentioned in the background art, the embodiments of the present invention provide a method for detecting genome damage after ultraviolet irradiation, and the method for detecting microorganism genome damage by using a dual-channel TaqMan probe fluorescent quantitative PCR technology has strong specificity and high sensitivity, and can be used for rapidly detecting microorganism genome damage after ultraviolet irradiation.
A method for detecting genome damage after ultraviolet irradiation detects the genome damage of microorganisms by a double-channel TaqMan probe fluorescent quantitative PCR technology, and comprises the following reaction steps:
step 4, respectively measuring quantitative PCR templates of the first control group in the step 3, dividing the PCR templates into a plurality of subgroups with the same volume, and respectively adding the subgroups into independent PCR amplification reaction systems; simultaneously amplifying long fragments and short fragments in any PCR amplification reaction system added with the template of the first control group; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; the reaction products of the PCR amplification reaction systems of all the obtained subgroups jointly form the first control group;
step 5, respectively measuring quantitative PCR templates of the first experimental group in the step 3, dividing the PCR templates into a plurality of subgroups with the same volume, and respectively adding the subgroups into independent PCR amplification reaction systems; simultaneously amplifying long fragments and short fragments in any PCR amplification reaction system added with the template of the first experimental group; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; the reaction products of the PCR amplification reaction systems of all the obtained subgroups jointly form the first experimental group;
step 6, collecting the fluorescence signal CT value of each subgroup in the first control group and the first experiment group by using the dual-channel probe;
step 7, calculating the CT difference value of each subgroup of the first control group by delta CTCK = CT length-CT length; the CT difference value calculation method of each subgroup of the first experimental group is that delta CTuv = CT length-CT length; judging the damage degree of the genomic DNA of the microorganisms in each subgroup of the first experimental group according to the delta CT = delta CTuv-delta CTCK-; wherein the Δ CTCK-is the average of the values of the subgroups of the first control group.
The lysis mix contained 10mM Tris-HCl pH =8.0 and 1% Triton X-100.
The first probe specifically acts on the fragment amplified by the short-fragment primer pair P1; the second probe specifically acts on the fragment amplified by the long-fragment primer pair P2.
When the delta CT is larger than or equal to L in the step 7, determining that the first experimental group microbial genome is damaged; determining that the first experimental group of microbial genomes are not damaged when Δ Δ CT < L.
L is the detection limit L ═ K ═ s)/b, b is the lowest response value/cycle number, K is the confidence coefficient, and s is the standard deviation for each subgroup in the second control group.
The standard deviation s determining step:
A. quantitatively obtaining the supernatant of the first culture solution obtained in the step 3 as a template of a dual-channel TaqMan probe fluorescent quantitative PCR of a second control group, and dividing the supernatant into a plurality of subgroups with the same volume to be respectively added into an independent PCR amplification reaction system; any PCR amplification reaction system added with the first culture solution supernatant is simultaneously used for amplifying long fragments and short fragments; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; a, the reaction products of each subgroup of PCR amplification reaction system form a second control group together;
B. acquiring the fluorescence signal CT value of each subgroup in the second control group by using the two-channel probe;
C. calculating a standard deviation s according to the delta CTCK values of all subgroups of the second control group; the calculation method of Δ CTCK of each subgroup of the second control group is Δ CTCK = CT long-CT short.
The number of the second control group subgroups is more than or equal to 20.
The number of the first control group subgroups is more than or equal to 3; the number of the first experimental group subgroups is more than or equal to 3.
The PCR amplification step of the invention:
a. adding the PCR amplification reaction system into a corresponding PCR reaction template, and respectively and independently operating for 5min at the temperature of 95 ℃;
b. after the operation a is finished, sequentially operating the product at the temperature of 95 ℃ for 10 seconds, operating the product at the temperature of 60 ℃ for 30 seconds and operating the product at the temperature of 72 ℃ for 40 seconds;
c. repeating the operation in the step b for 35 times to obtain the required PCR amplification reaction product.
The beneficial effects include:
the method for detecting the microbial genome damage by using the dual-channel TaqMan probe fluorescent quantitative PCR technology has stronger specificity and higher sensitivity, and can be used for quickly detecting the identification of the microbial genome damage after ultraviolet irradiation.
Specifically, the difference value delta Delta CT of CT values before and after ultraviolet irradiation of two fragments is obtained by amplification of long and short fragments, and the damage condition of the microbial genome subjected to ultraviolet irradiation can be directly detected; from the beginning of ultraviolet irradiation, the whole time flow only needs 1-2 hours, compared with other methods for detecting DNA damage, the detection time is greatly reduced, and the purpose of rapid detection is achieved; different long and short fragments are amplified in the same reaction tube by using a dual-channel probe method, so that errors caused by sample adding are eliminated, and the specificity and the precision of an experiment are improved compared with a dye method.
Drawings
FIG. 1 is a schematic flow diagram of an experimental procedure;
FIG. 2 is an amplification curve of different fragments of E.coli K2 in example 3;
FIG. 3 is a graph showing the difference between E.coli K2 Δ Δ CT in example 3;
FIG. 4 is a graph showing the amplification curves of different fragments of E.coli ATCC 25922 in example 4;
FIG. 5 is a graph showing the difference in E.coli ATCC 25922 Δ Δ CT in example 4;
FIG. 6 is a graph showing the amplification curves of different fragments of Staphylococcus aureus in example 5;
FIG. 7 is a Δ Δ CT difference chart of Staphylococcus aureus in example 5.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of the present invention.
The definition of "detection limit" in appendix a of GB/T5009.1-2003 general rules of food hygiene test methods, wherein the formula L ═ K × s)/b (L is the detection limit, b is the lowest response value/cycle number, and the confidence coefficient K, s is the standard deviation of n blank samples) is used to calculate the detection limit L of the identification method.
The test uses a certain type of fluorescent quantitative PCR detection equipment, and the confidence coefficient K is 0.99, b =1, and L =0.99 s.
As shown in figure 1, the method for detecting genome damage after ultraviolet irradiation detects the genome damage of microorganisms by a double-channel TaqMan probe fluorescence detection quantitative PCR technology on DNA of the microorganisms, and comprises the following preparation steps:
(1) culturing with corresponding culture medium to obtain required microbial culture solution;
(2) quantitatively obtaining a certain volume of the microbial culture solution as a first culture solution without additional treatment; quantitatively obtaining a certain volume of the microbial culture solution as a second culture solution, and irradiating for a period of time under certain ultraviolet intensity;
(3) adding a lysis raw material into the first culture solution and the second culture solution respectively to prepare a lysis mixed solution, performing lysis for 3-10min respectively, and centrifuging at the rotation speed of 6000-10000 rpm for 5-10 min to obtain supernatants of the first control group and the first experiment group, wherein the supernatants are used as templates of the dual-channel TaqMan probe fluorescence quantitative PCR of the first control group and the first experiment group respectively;
(4) respectively and quantitatively weighing the control group templates in the step (3), dividing the control group templates into a plurality of subgroups with the same volume, and respectively adding the subgroups into independent PCR amplification reaction systems; the amplification of the long fragment and the short fragment is simultaneously carried out in any PCR amplification reaction system added with the template of the PCR; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; (4) the reaction products of the PCR amplification reaction systems of all subgroups form a first control group together;
(5) respectively and quantitatively weighing the first experimental group template in the step (3), dividing the first experimental group template into a plurality of subgroups with the same volume, and respectively adding the subgroups into independent PCR amplification reaction systems; the amplification of the long fragment and the short fragment is simultaneously carried out in any PCR amplification reaction system added with the template of the PCR; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; (5) the reaction products of the PCR amplification reaction systems of all subgroups jointly form a first experimental group;
(6) quantitatively obtaining a template of the first control group dual-channel TaqMan probe fluorescent quantitative PCR in the step (3), dividing the template into a plurality of subgroups with the same volume and respectively adding the subgroups into independent PCR amplification reaction systems; the amplification of the long fragment and the short fragment is simultaneously carried out in any PCR amplification reaction system added with the template of the PCR; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; (6) the reaction products of the PCR amplification reaction systems of all subgroups form a second control group together;
(7) acquiring the CT values of the fluorescence signals of each subgroup in the first control group, the second control group and the first experimental group by using the dual-channel probe;
(8) performing double-channel TaqMan probe fluorescent quantitative PCR reaction on each subgroup of the second control group, and calculating delta CTCK values of each subgroup of the second control group to obtain a standard deviation s; the calculation method of the delta CTCK of each subgroup of the second control group is that the delta CTCK = CT length-CT length;
(9) the CT difference value calculation method of each subgroup of the first control group is delta CTCK = CT length-CT length; the CT difference value calculation method of each subgroup of the first experimental group is that delta CTuv = CT length-CT length; and judging the damage degree of the genomic DNA of the microorganisms in each subgroup of the first experimental group according to the value of delta CT = delta CTuv-delta CTCK-, wherein the value of delta CT is in direct proportion to the damage degree of the genomic DNA of the microorganisms within a set numerical range. Wherein Δ CTCK-is the average of the calculated values for each subgroup of the first control group; when the delta CT is larger than or equal to L, the first experimental group microbial genome is damaged; Δ Δ CT < L the first experimental group of microbial genomes were not damaged; and L is a detection limit L ═ K x s)/b, b is the lowest response value/cycle number, K is a confidence coefficient, and s is the standard deviation of each subgroup of the second control group.
The ultraviolet intensity of the ultraviolet treatment is 10-30000uw/cm 2; the irradiation time is 0.5-100 seconds.
The number of the first control group subgroups is more than or equal to 3; the number of the first experiment group is more than or equal to 3; the number of the second control group subgroups is more than or equal to 20.
The lysis mix contained 10mM Tris-HCl pH =8.0 and 1% Triton X-100.
The first probe specifically acts on the fragment amplified by the short-fragment primer pair P1; the second probe specifically acts on the fragment amplified by the short-fragment primer pair P2.
The PCR amplification step comprises:
a. adding the PCR amplification reaction systems of the subgroups in the first control group, the subgroups in the second control group and the subgroups in the first experimental group into corresponding PCR reaction templates, and respectively and independently operating for 5min at the temperature of 95 ℃;
b. after the operation a is finished, sequentially operating the product at the temperature of 95 ℃ for 10 seconds, operating the product at the temperature of 60 ℃ for 30 seconds and operating the product at the temperature of 72 ℃ for 40 seconds;
c. repeating the operation in the step b for 35 times to obtain the required PCR amplification reaction product.
In order to better verify the microbial genome damage method disclosed by the invention, the invention also discloses and introduces a second experimental group, particularly as the experimental group which is not irradiated by ultraviolet light and has genome damage, the rationality of the method and the criterion is verified, whether the delta CT is less than L or not is judged by substituting the second experimental group into the corresponding criterion, and particularly, the preparation method of the second experimental group is completely the same as that of the second control group; the number of the second experimental group subgroups is more than or equal to 3. The CT difference value calculation method of the second experimental group is delta CTck' = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTck' -delta CTCK-; the above-mentioned Δ CTCK-is the average value of the calculated values of the subgroups of the first control group, and Δ CTck' is the measured value of the subgroups of the second experimental group.
Example 1
On the basis of the disclosed embodiments, the present invention preferably discloses an embodiment of a method for detecting genome damage after ultraviolet irradiation, which is specifically performed as follows:
(1) culturing with corresponding culture medium to obtain required microbial culture solution;
(2) quantitatively obtaining a certain volume of the microbial culture solution as a culture solution raw material of a first control group, a second control group and a second experimental group without additional treatment; quantitatively obtaining a certain volume of the microbial culture solution as a culture solution raw material of a first experimental group, and irradiating for a period of time under certain ultraviolet intensity; wherein the second control group is used to determine the limit of detection and the first control group and the first experimental group are used to verify the validity of the DNA damage detection method;
(3) adding a cracking raw material into the microbial culture solution to prepare a cracking mixed solution, respectively cracking for 3-10min, and centrifuging at the rotation speed of 7000-10000rpm for 6-10min to obtain supernatants of the first control group, the second experimental group and each subgroup of the first experimental group as a fluorescent quantitative PCR template;
(4) measuring quantitative templates of each subgroup of the second control group, the third control group, the second experiment group and the first experiment group in the step (3), respectively adding the templates into a PCR amplification reaction system, and respectively carrying out long-short fragment PCR amplification on each subgroup; the PCR amplification reaction system also comprises Taq buffer with 10X concentration, Mg2+ salt with 2-4mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water;
(5) acquiring the CT value of the fluorescence signal of each subgroup of the second control group, the third control group, the second experimental group and the first experimental group by using the dual-channel probe;
(6) obtaining a detection limit of a detection method, wherein the method is operated by carrying out the same fluorescent quantitative PCR reaction by using the second control group to obtain a standard deviation s of delta CTCK; the CT difference value of the second control group is calculated by delta CTCK = CT length-CT length;
(7) the CT difference value of the first control group is calculated by delta CTCK = CT length-CT length; the CT difference value of the first experimental group is calculated by delta CTuv = CT length-CT length; judging whether the genome of the first experimental group of microorganisms is damaged by ultraviolet irradiation according to the delta CT = delta CTuv-delta CTCK-, wherein the value of the delta CT is in direct proportion to the damage degree of the genome DNA of the microorganisms within a set value range; the CT difference value calculation method of the second experimental group is delta CTck' = CT length-CT length; judging whether the genome of the second experimental group of microorganisms is damaged by ultraviolet irradiation according to the delta CT = delta CTck' -delta CTCK-; the delta CTCK-is the average value of the calculated values of all subgroups of the second control group, and the delta CTck' is the measured value of all subgroups of the second experimental group;
(8) the second experimental group and the first experimental group are judged according to the following method, and when the ultraviolet irradiation damage judgment method is that delta CT is larger than or equal to L, the microbial genome of the experimental group is damaged; the genome of the experimental group of microorganisms was not damaged by Δ Δ CT < L.
The ultraviolet intensity of the ultraviolet treatment is 100-10000uw/cm 2; the irradiation time is 0.5-80 seconds.
The number of the first control group subgroups is more than or equal to 3; the number of the first experiment group and the second experiment group is more than or equal to 3; the number of the second control group subgroups is more than or equal to 20.
The lysis mix contained 10mM Tris-HCl pH =8.0 and 1% Triton X-100.
Based on the published method, the identification method detection limit L =0.99s is calculated from the detection limit formula L ═ (K ×) b (L is the detection limit, the lowest response value b =1(/ cycle number), the confidence coefficient K ═ 0.99, s is the standard deviation of n blanks).
This example also discloses an embodiment of a PCR amplification method:
a. respectively adding extracts of cracked microbial culture solutions as templates into a PCR amplification reaction system, and simultaneously respectively carrying out long-fragment and short-fragment PCR amplification; the PCR amplification reaction system runs for 5min at the temperature of 95 ℃;
b. after the operation a is finished, sequentially operating the product at the temperature of 95 ℃ for 10 seconds, operating the product at the temperature of 60 ℃ for 30 seconds and operating the product at the temperature of 72 ℃ for 40 seconds;
c. repeating the operation in the step b for 20-40 times to obtain the required PCR amplification reaction product.
In some alternative embodiments, the second experimental set of three sub-set samples and the first experimental set of three sub-set samples were used as unknown radiation damage samples for damage determination using the methods disclosed above: the calculated delta-delta CT values of the second experimental group are all less than 0.99 times of s; the calculated Δ CT values for the first experimental group were all greater than 0.99 times s.
Obviously, the experimental result is within the theoretical prediction range, and the method for detecting the microbial genome damage by using the PCR technology is proved to have the advantages of stronger specificity and higher sensitivity, and can be used for rapidly detecting the identification and the like of the microbial genome damage after ultraviolet irradiation.
Example 2
On the basis of the disclosed embodiments, the present invention preferably discloses an embodiment of a method for detecting genome damage after ultraviolet irradiation, which is specifically performed as follows:
(1) culturing with corresponding culture medium to obtain required microbial culture solution;
(2) quantitatively obtaining a certain volume of the microbial culture solution as a culture solution raw material of a first control group, a second control group and a second experimental group without additional treatment; quantitatively obtaining a certain volume of the microbial culture solution as a culture solution raw material of a first experimental group, and irradiating for a period of time under certain ultraviolet intensity; wherein the second control group is used to determine the limit of detection and the first control group, the second experimental group and the first experimental group are used to verify the rationality of the DNA damage detection method;
(3) adding lysis raw materials into the control group and experimental group microbial culture solution to prepare lysis mixed solution, respectively lysing for 3-10min, and centrifuging at 7000-10000rpm for 6-10min to obtain supernatants of the second control group, the third control group, the second experimental group and the first experimental group as fluorescent quantitative PCR templates;
(4) quantitatively weighing templates of each subgroup of the second control group, the third control group, the second experimental group and the first experimental group in the step (3), respectively adding the templates into a PCR amplification reaction system, and respectively carrying out long-short fragment PCR amplification on each subgroup; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 3-4mM, a first probe with 0.2-0.3uM, a second probe with 0.2-0.3uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.3-0.4uM, a long segment primer pair P2 with 0.2-0.3uM, a proper amount of DNA polymerase and sterilized water;
(5) acquiring fluorescence signal CT values of each subgroup of the second control group, the third control group, the second experimental group and the first experimental group by using the dual-channel probe;
(6) obtaining a detection limit of a detection method, wherein the method is operated by carrying out the same fluorescent quantitative PCR reaction by using the second control group to obtain a standard deviation s of delta CTCK; the CT difference value of the second control group is calculated by delta CTCK = CT length-CT length;
(7) the CT difference value of the first control group is calculated by delta CTCK = CT length-CT length; the CT difference value of the first experimental group is calculated by delta CTuv = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTuv-delta CTCK-, wherein the value of the delta CT is in direct proportion to the damage degree of the microbial genome DNA within a set value range; the CT difference value calculation method of the second experimental group is delta CTck' = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTck' -delta CTCK-; the delta CTCK-is the average value of the calculated values of all subgroups of the second control group, and the delta CTck' is the measured value of all subgroups of the second experimental group;
(8) the second experimental group and the first experimental group are judged according to the following method, and when the ultraviolet irradiation damage judgment method is that delta CT is more than or equal to 0.99s, the microbial genome of the experimental groups is damaged; the genome of the experimental group of microorganisms was not damaged by Δ Δ CT <0.99 s.
The ultraviolet intensity of the ultraviolet treatment is 1000-10000uw/cm 2; the irradiation time is 1-80 seconds.
The number of the first control group subgroups is more than or equal to 3; the number of the first experiment group and the second experiment group is more than or equal to 3; the number of the second control group subgroups is more than or equal to 20.
The lysis mix contained 10mM Tris-HCl pH =8.0 and 1% Triton X-100.
Based on the published method, the identification method detection limit L =0.99s is calculated from the detection limit formula L ═ (K ×) b (L is the detection limit, the lowest response value b =1(/ cycle number), the confidence coefficient K ═ 0.99, s is the standard deviation of n blanks).
This example also discloses an embodiment of a PCR amplification method:
a. respectively adding the microbial culture solution into a PCR amplification reaction system by taking a cracked extract as a template, and simultaneously respectively carrying out long-short fragment PCR amplification; the PCR amplification reaction system runs for 5min at the temperature of 95 ℃;
b. after the operation a is finished, sequentially operating the product at the temperature of 95 ℃ for 10 seconds, operating the product at the temperature of 60 ℃ for 30 seconds and operating the product at the temperature of 72 ℃ for 40 seconds;
c. repeating the operation in the step b for 20-30 times to obtain the required PCR amplification reaction product.
In some alternative embodiments, the second experimental set of three sub-set samples and the first experimental set of three sub-set samples were used as unknown radiation damage samples for damage determination using the methods disclosed above: the calculated delta-delta CT values of the second experimental group are all less than 0.99 times of s; the calculated Δ CT values for the first experimental group were all greater than 0.99 times s.
Obviously, the experimental result is within the theoretical prediction range, and the method for detecting the microbial genome damage by using the PCR technology is proved to have the advantages of stronger specificity and higher sensitivity, and can be used for rapidly detecting the identification and the like of the microbial genome damage after ultraviolet irradiation.
Example 3
On the basis of the disclosed embodiments, the invention discloses an embodiment of a method for detecting genome damage after ultraviolet irradiation, which specifically comprises the following operations:
(1) culturing with LB culture medium to obtain Escherichia coli K12 (0D: 1.0-2.0);
(2) taking 5 parts of 50 ul of bacterial liquid, and taking three parts of the bacterial liquid as a first control group, a second control group and a second experimental group respectively without treatment; two parts are taken as experimental groups, and are irradiated for 1 second and 2 seconds by ultraviolet under the condition that the ultraviolet intensity is 3000 uw/cm 2;
(3) cracking with lysis solution for 3 min; the lysate contains 10mM Tris-HCL at pH =8.0 and 1% Triton X-100;
(4) respectively centrifuging at 6000 rpm for 5 minutes to obtain supernatants which are respectively used as templates of fluorescent quantitative PCR;
(5) adding the template in the step (4) into a PCR reaction system, amplifying long and short fragments simultaneously, collecting a dual-channel probe fluorescent signal, and repeating each reaction for three times;
first probe sequence: 5' -6-FAM-CTGCTCACCGACAAAGCTGATCTCT-3 ' BHQ1'
A second probe sequence: 5' -6-VIC-CCTGAAAGCCCAACAGGAAATAACCTCG-3 ' BHQ1'
Short segment primer pair sequences: 5'-GCATTATCCAGCACGTTG-3'
R:5'-GCAAAGGGGTCAATATCTCTC-3'
Long fragment primer pair sequences: 5'-ATGTACTTGCTCAGCCGTC-3'
R:5'- ACCTGATGTACGTAATAAACCGT-3'
(6) As shown in FIG. 2, dual-channel amplification results (the solid line is a short-segment amplification curve; the dotted line is a long-segment amplification curve) under different ultraviolet irradiation time conditions are obtained; the CT difference value of the first control group is calculated by delta CTCK = CT length-CT length; the CT difference value calculation method of the experimental group is that delta CTuv = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTuv-delta CTCK; the CT difference value calculation method of the second experimental group is delta CTck' = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTck' -delta CTCK-; the delta CTCK-is the average value of the calculated values of the subgroups of the second control group, the delta CTck' is the measured value of the subgroups of the second experimental group, and the change of the damage delta CT of the DNA caused by ultraviolet rays along with the ultraviolet irradiation time is shown in FIG. 3;
(7) obtaining the detection limit of the detection method, wherein the standard deviation of the control group is 0.99 times that of the control group, and performing the same fluorescent quantitative PCR reaction for 20 times by using a second control group which is not subjected to ultraviolet irradiation according to the method to obtain the standard deviation of the delta CTCK result of the second control group of 0.239;
(8) judging whether the genome DNA is damaged by ultraviolet irradiation according to the first control group, the second experimental group and the experimental group delta Delta CT, wherein the delta Delta CT is less than 0.237, and the DNA is not damaged; delta CT is more than or equal to 0.237, and DNA is damaged.
The experimental results show that the second experimental group of three samples and the experimental group of three samples were used as unknown radiation damage samples for damage assessment using the method disclosed above:
calculated delta CT values for the second experimental group were 0.12, 0.14 and 0.11;
calculated delta CT for experimental group is 1 s: 1.9, 1.93 and 1.88; 2 s: 3.02, 2.98 and 3.03.
Obviously, the experimental result is within the theoretical prediction range, and the method for detecting the microbial genome damage by using the PCR technology is proved to have the advantages of stronger specificity and higher sensitivity, and can be used for rapidly detecting the identification and the like of the microbial genome damage after ultraviolet irradiation.
Example 4
On the basis of the disclosed embodiments, the invention discloses an embodiment of a method for detecting genome damage after ultraviolet irradiation, which specifically comprises the following operations:
(1) obtaining ATCC 25922 (0D: 1.0-2.0) by culturing in LB medium;
(2) taking 5 parts of 50 ul of bacterial liquid, and taking three parts of the bacterial liquid as a first control group, a second control group and a second experimental group respectively without treatment; two parts are taken as experimental groups, and are irradiated for 1 second and 2 seconds by ultraviolet under the condition that the ultraviolet intensity is 3000 uw/cm 2;
(3) cracking with lysis solution for 3 min; the lysate contains 10mM Tris-HCL at pH =8.0 and 1% Triton X-100;
(4) respectively centrifuging at 8000 rpm for 5 minutes to obtain supernatants which are respectively used as templates of fluorescent quantitative PCR;
(5) adding the template in the step (4) into a PCR reaction system, amplifying long and short fragments simultaneously, collecting a dual-channel probe fluorescent signal, and repeating each reaction for three times;
first probe sequence: 5 '6-FAM-CTGCTCACCAACAAAGCTGATCTCT-3' BHQ1
A second probe sequence: 5' -6-VIC-ACCCTGACCGATCTTCTCACC-3 ' BHQ1'
Short segment primer pair sequences: 5'-GTATCAACGCAAAGGGGTC-3'
R:5'-AGGCATTATCCAGCACATTAC-3'
Long fragment primer pair sequences: 5'-TATCTTTCAGCCAGGCAGTC-3'
R:5'-AACGCCAACACCATCTTC-3'
(6) As shown in FIG. 4, dual-channel amplification results (the solid line is a short-segment amplification curve; the dotted line is a long-segment amplification curve) under different ultraviolet irradiation time conditions were obtained; the CT difference value of the first control group is calculated by delta CTCK = CT length-CT length; the CT difference value calculation method of the experimental group is that delta CTuv = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTuv-delta CTCK; the CT difference value calculation method of the second experimental group is delta CTck' = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTck' -delta CTCK-; the delta CTCK-is the average value of the calculated values of the subgroups of the second control group, the delta CTck' is the measured value of the subgroups of the second experimental group, and the change of the damage delta CT of the DNA caused by ultraviolet rays along with the ultraviolet irradiation time is shown in FIG. 5;
(7) obtaining the detection limit of the detection method, wherein the detection limit is 3 times of the standard deviation of a control group, and performing the same fluorescent quantitative PCR reaction for 20 times by using a second control group which is not subjected to ultraviolet irradiation according to the method to obtain the standard deviation of the delta CTCK result of the second control group, wherein the standard deviation of the delta CTCK result of the second control group is 0.225;
(8) judging whether the genome DNA is damaged by ultraviolet irradiation according to the first control group, the second experimental group and the experimental group delta Delta CT, wherein the delta Delta CT is less than 0.223, and the DNA is not damaged; delta CT is not less than 0.223, and DNA is damaged.
The experimental results show that the second experimental group of three samples and the experimental group of three samples were used as unknown radiation damage samples for damage assessment using the method disclosed above:
the calculated Δ CT values for the second experimental group were 0.07, 0.13 and 0.09;
calculated delta CT for experimental group is 1 s: 1.14, 1.1, 1.16, 2 s: 1.81, 1.79, 1.81.
Obviously, the experimental result is within the theoretical prediction range, and the method for detecting the microbial genome damage by using the PCR technology is proved to have the advantages of stronger specificity and higher sensitivity, and can be used for rapidly detecting the identification and the like of the microbial genome damage after ultraviolet irradiation.
Example 5
On the basis of the disclosed embodiments, the invention discloses an embodiment of a method for detecting genome damage after ultraviolet irradiation, which specifically comprises the following operations:
(1) culturing with broth to obtain Staphylococcus aureus (0D: 1.0-2.0);
(2) taking 5 parts of 50 ul of bacterial liquid, and taking three parts of the bacterial liquid as a first control group, a second control group and a second experimental group respectively without treatment; two parts are taken as experimental groups, and are irradiated for 1 second and 2 seconds by ultraviolet under the condition that the ultraviolet intensity is 3000 uw/cm 2;
(3) cracking with lysis solution for 5 min; the lysate contains 10mM Tris-HCL at pH =8.0 and 1% Triton X-100;
(4) respectively centrifuging at 6000 rpm for 5 minutes to obtain supernatants which are respectively used as templates of fluorescent quantitative PCR;
(5) adding the template in the step (4) into a PCR reaction system, amplifying long and short fragments simultaneously, collecting a dual-channel probe fluorescent signal, and repeating each reaction for three times;
first probe sequence: 5 '6-FAM-CCGATATCACCGATACCACTACCATC-3' BHQ 1;
a second probe sequence: 5 'HEX-CGCACATCAGATGCCACACATCTA-3' BHQ1
Short segment primer pair sequences: 5'-CTTCAGATAATAGCTTACGGTCTC-3'
5'- GTAAAGCTGGTCGCATTC -3'
Long fragment primer pair sequences: 5'-CCTCTGCATAGCCATATGTTG-3'
5'- GACACCGCATTGTTGCTT -3'
(6) As shown in FIG. 6, dual-channel amplification results (the solid line is a short-segment amplification curve; the dotted line is a long-segment amplification curve) under different UV irradiation time conditions were obtained; the CT difference value of the first control group is calculated by delta CTCK = CT length-CT length; the CT difference value calculation method of the experimental group is that delta CTuv = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTuv-delta CTCK; the CT difference value calculation method of the second experimental group is delta CTck' = CT length-CT length; judging whether the microbial genome DNA is damaged by ultraviolet irradiation according to the delta CT = delta CTck' -delta CTCK-; the delta CTCK-is the average value of the calculated values of the subgroups of the second control group, the delta CTck' is the measured value of the subgroups of the second experimental group, and the change of the damage delta CT of the DNA caused by ultraviolet rays along with the ultraviolet irradiation time is shown in FIG. 7;
(7) obtaining the detection limit of the detection method, wherein the standard deviation of the control group is 0.99 times that of the control group, and performing the same fluorescent quantitative PCR reaction for 20 times by using a second control group which is not subjected to ultraviolet irradiation according to the method to obtain the standard deviation of the delta CTCK result of the second control group, wherein the standard deviation of the delta CTCK result of the second control group is 0.215;
(8) judging whether the genome DNA is damaged by ultraviolet irradiation according to the first control group, the second experimental group and the experimental group delta Delta CT, wherein the delta Delta CT is less than 0.213, and the DNA is not damaged; delta CT is not less than 0.213, and DNA is damaged.
The experimental results show that the second experimental group of three samples and the experimental group of three samples were used as unknown radiation damage samples for damage assessment using the method disclosed above:
calculated Δ CT values for the second experimental group were 0.152, 0.146 and 0.151;
calculated delta CT for experimental group is 1 s: 1.88, 1.92, 1.89 and 2 s: 3.26,3.39,3.56.
Obviously, the experimental result is within the theoretical prediction range, and the method for detecting the microbial genome damage by using the PCR technology is proved to have the advantages of stronger specificity and higher sensitivity, and can be used for rapidly detecting the identification and the like of the microbial genome damage after ultraviolet irradiation.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.
Claims (9)
1. A method for detecting genome damage after ultraviolet irradiation is characterized in that the method detects the genome damage of microorganisms by a double-channel TaqMan probe fluorescent quantitative PCR technology, and comprises the following reaction steps:
step 1, culturing by using a corresponding culture medium to obtain a required microbial culture solution;
step 2, quantitatively obtaining a certain volume of the microbial culture solution as a first culture solution without additional treatment; quantitatively obtaining a certain volume of the microbial culture solution as a second culture solution, and irradiating for a period of time under certain ultraviolet intensity;
step 3, adding cracking raw materials into the first culture solution and the second culture solution respectively to prepare cracking mixed solutions, cracking for 3-10min respectively, and centrifuging for 5-10 min at the rotating speed of 6000-10000 rpm to obtain supernatants of the first culture solution and the second culture solution; the supernatant of the first culture solution is used as a template of a first control group dual-channel TaqMan probe fluorescent quantitative PCR; the second culture solution supernatant is used as a template of the dual-channel TaqMan probe fluorescent quantitative PCR of the first experiment group;
step 4, respectively measuring quantitative PCR templates of the first control group in the step 3, dividing the PCR templates into a plurality of subgroups with the same volume, and respectively adding the subgroups into independent PCR amplification reaction systems; simultaneously amplifying long fragments and short fragments in any PCR amplification reaction system added with the template of the first control group; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; the reaction products of the PCR amplification reaction systems of all the obtained subgroups jointly form the first control group; the length of the short fragment is 60-300 bp; the length of the long fragment is 1000-2500 bp;
step 5, respectively measuring quantitative PCR templates of the first experimental group in the step 3, dividing the PCR templates into a plurality of subgroups with the same volume, and respectively adding the subgroups into independent PCR amplification reaction systems; simultaneously amplifying long fragments and short fragments in any PCR amplification reaction system added with the template of the first experimental group; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; the reaction products of the PCR amplification reaction systems of all the obtained subgroups jointly form the first experimental group;
step 6, collecting the fluorescence signal CT value of each subgroup in the first control group and the first experiment group by using the dual-channel probe;
step 7, calculating the CT difference value of each subgroup of the first control group by delta CTCK = CT length-CT length; the CT difference value calculation method of each subgroup of the first experimental group is that delta CTuv = CT length-CT length; judging the damage degree of the genomic DNA of the microorganisms in each subgroup of the first experimental group according to the delta CT = delta CTuv-delta CTCK-; wherein the Δ CTCK-is the average of the values of the subgroups of the first control group.
2. The method for detecting genome damage after ultraviolet irradiation according to claim 1, wherein the ultraviolet irradiation intensity in step 2 is 10-30000uw/cm 2; the irradiation time is 0.5-100 seconds.
3. The method of claim 1, wherein the lysis cocktail comprises 10mM ph =8.0 Tris-HCL and 1% Triton X-100.
4. The method for detecting genome damage after ultraviolet irradiation of claim 1, wherein the first probe specifically acts on the amplified fragment of the short-fragment primer pair P1; the second probe specifically acts on the fragment amplified by the long-fragment primer pair P2.
5. The method for detecting genome damage after ultraviolet irradiation according to claim 1, further comprising: when the delta CT is larger than or equal to L in the step 7, determining that the first experimental group microbial genome is damaged; determining that the first experimental group of microbial genomes are not damaged when Δ Δ CT < L; l is the detection limit L ═ K ═ s)/b, b is the lowest response value/cycle number, K is the confidence coefficient, and s is the standard deviation for each subgroup in the second control group.
6. The method for detecting genome damage after ultraviolet irradiation according to claim 5, further comprising: determining the standard deviation s of each subgroup of the second control group by:
A. quantitatively obtaining the supernatant of the first culture solution obtained in the step 3 as a template of a dual-channel TaqMan probe fluorescent quantitative PCR of a second control group, and dividing the supernatant into a plurality of subgroups with the same volume to be respectively added into an independent PCR amplification reaction system; any PCR amplification reaction system added with the first culture solution supernatant is simultaneously used for amplifying long fragments and short fragments; the PCR amplification reaction system comprises Taq buffer with 10X concentration, Mg2+ salt with 1-5mM, a first probe with 0.2-0.4uM, a second probe with 0.2-0.4uM, dNTPs with 0.2mM, a short segment primer pair P1 with 0.2-0.4uM, a long segment primer pair P2 with 0.2-0.4uM, a proper amount of DNA polymerase and sterilized water; a, the reaction products of each subgroup of PCR amplification reaction system form a second control group together;
B. acquiring the fluorescence signal CT value of each subgroup in the second control group by using the two-channel probe;
C. calculating a standard deviation s according to the delta CTCK values of all subgroups of the second control group; the calculation method of Δ CTCK of each subgroup of the second control group is Δ CTCK = CT long-CT short.
7. The method for detecting genome damage after ultraviolet irradiation of claim 6, wherein the number of the second control group subgroups is not less than 20.
8. The method for detecting genome damage after ultraviolet irradiation according to claim 1, wherein the number of the first control group subgroups is not less than 3; the number of the first experimental group subgroups is more than or equal to 3.
9. The method for detecting genome damage after ultraviolet irradiation according to any one of claims 1 to 8, comprising the PCR amplification step of:
a. adding the PCR amplification reaction system into a corresponding PCR reaction template, and respectively and independently operating for 5min at the temperature of 95 ℃;
b. after the operation a is finished, sequentially operating the product at the temperature of 95 ℃ for 10 seconds, operating the product at the temperature of 60 ℃ for 30 seconds and operating the product at the temperature of 72 ℃ for 40 seconds;
c. repeating the operation in the step b for 35 times to obtain the required PCR amplification reaction product.
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