CN113430228A - Preparation and detection method of biological indicator for detecting disinfection and sterilization - Google Patents
Preparation and detection method of biological indicator for detecting disinfection and sterilization Download PDFInfo
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Abstract
The invention discloses a preparation and detection method of a biological indicator for detecting disinfection and sterilization, wherein the biological indicator virus is a pseudovirus particle obtained by modifying and deleting a gene sequence of an encoding envelope protein in a euvirus and co-transfecting a 293T cell line with envelope glycoprotein genes of other viruses. One of the biological indicator virus and the indicator cell line carries a luciferase reporter gene. After the biological indicator virus is used for infecting the indicating cell line, luciferase gene expression is up-regulated, and then the infection and replication efficiency of the biological indicator virus is judged by detecting the expression condition of luciferase. The biological indicator virus can show the inactivation rate in a gradient form after absorbing ozone, ultraviolet rays and cobalt sources with different doses and being irradiated by an accelerator. The biological indicator virus based on the luciferase has the advantages of simple preparation, reliable result and the like, and is convenient and rapid to use, high in safety, strong in operability and high in sensitivity.
Description
Technical Field
The invention relates to the technical field of biological monitoring in disinfection and sterilization, in particular to a preparation and detection method of a biological indicator for detecting disinfection and sterilization.
Background
For example, Wang Ping Liang, Zhang Gao hong, Zheng Yongtang in the article "research progress for screening antiviral drugs based on pseudoviruses" mentioned that pseudoviruses are a kind of viruses with exogenous viral envelope formed by integrating envelope glycoproteins of other viruses, and the self genome still maintains the characteristics of the original viral genome. Pseudoviruses have the advantage of high safety because they lose the self-replication ability of the virus, and have been widely used for the study of viruses such as HIV, H5N1, and HCV. The final phrase is also given in the article: in the past decades, the importance of pseudoviral technology in basic virology research, gene transduction, neutralizing antibody detection and therapeutic applications has been established, which will increasingly be applied in basic research and clinical therapy, especially in the research of some highly pathogenic viruses. Pseudoviruses are also a hot research focus in recent years in vaccine construction strategies and gene therapy research. For the research field of antiviral drugs, a pseudovirus screening platform also plays an increasingly important role, and it is believed that with the continuous optimization of a functional system, the pseudovirus system will realize standardization in antiviral drug screening. The pseudovirus technology has many disadvantages, because the infection and replication processes are not completely the same, the pseudovirus cannot completely replace the live virus, the active substance after screening the pseudovirus still needs to use the live virus to confirm the activity, and scientists are working on the improvement of the pseudovirus system. With the development of molecular biology and medicine, pseudovirus technology will be more mature and perfected, and the application field will be wider and wider.
From the above article, we can clearly see that the research position of pseudoviruses in virology is in an increasingly important position, and we should also make intensive development on the concept of pseudoviruses to the greatest extent. The continuous application of the pseudoviruses is believed to lead us to continuously enter the mechanism of the viruses, so that various viruses with complicated and numerous varieties in the current world form can be better dealt with, and a firm foundation is laid for the long-term stable development of human beings.
Against the background, the invention provides a preparation and detection method of a biological indicator for detecting disinfection and sterilization.
Disclosure of Invention
The invention aims to provide a preparation method and a detection method of a biological indicator for detecting disinfection and sterilization, wherein the indicator has the advantages of simple preparation, reliable result and the like, and is convenient and quick to use, high in safety, strong in operability and high in sensitivity.
In order to achieve the purpose, the invention provides the following technical scheme: a method of making a biological indicator for detecting sterilization comprising the steps of:
s1, preparing transfection when 293T cells grow to 70-80%, wherein the mass ratio of the skeleton plasmid pSG 3-delta env to the envelope plasmid pcDNA3.1-env is 4: 1;
s2, after transfection for 4h, DMEM complete medium (containing 10% FBS) is replaced, and the virus is recovered after continuous culture for 48 h.
Preferably, the biological indicator for detecting sterilization is applied to ozone, ultraviolet rays, cobalt sources and accelerator sterilization work as a sterilization indicator. When applied, the following conditions should be satisfied: the conditions of ozone disinfection and sterilization are as follows: when the space humidity is more than 75%, the Concentration Time (CT) value is defined as the ozone gas concentration (ppm) multiplied by the administration duration (min), such as 0.05ppm for 20 hours or 0.1ppm for 10 hours; the conditions of ultraviolet disinfection and sterilization are as follows: is 20-30cm below the radiation source, and has radiation intensity of 0.1mW/cm2Duration 30 seconds, 60 seconds, 300 seconds; the conditions of cobalt source disinfection and sterilization are as follows: at a temperature of-78 ℃ to 10 ℃ using60Co gamma rays are irradiated by adopting the average dose rate of 0.4kGy/4.5h-3kGy/4.5h, and the total irradiation dose is 2kGy-13 kGy; the conditions for sterilizing the accelerator are as follows: the biological indicator virus is wrapped on a cell plate and dried in air, and after accelerator irradiation is carried out at room temperature (2-10 ℃), the adsorbed virus is liberated by using a complete culture medium and then detected.
Preferably, the biological indicator has an infectious titer of TCID50 of 10-4-10-5/mL。
Preferably, the biological indicator also comprises an indicating cell line corresponding to the biological indicator, and the indicating cell line is a susceptible cell line of the biological indicator.
Preferably, one of the biological indicator and the indicator cell line carries a luciferase reporter gene.
Preferably, a method for detecting the activity of a biological indicator for sterilization indication, comprising the steps of:
(1) to the indicated cell dilutions DEAE-Dextran was added at a final concentration of 15. mu.g/mL and the cells were treated at 1X 104The amount per well was added to a 96 well plate;
(2) sequentially diluting the biological indicator viruses by 5 times;
(3) detection using a luciferase substrate;
(4) for the calculation of TCID50, the Cutoff value was first calculated, and the Cutoff value was 3xC calr (cell control mean), and the number of positive and negative results were found from the fluorescence values, and TCID50/ml was obtained by Reed-Muench bikes method.
Compared with the prior art, the invention has the beneficial effects that:
(1) the biological indicator virus of the invention is combined with the prior ozone and ultraviolet sterilization biological indicator, namely bacillus subtilis and60compared with a Co irradiation sterilization biological indicator, namely bacillus pumilus, the Co irradiation sterilization biological indicator has higher similarity and higher safety with real viruses;
(2) the biological indicator virus detects the expression condition of luciferase by an enzyme-labeling instrument, so that the result is more accurate and the sensitivity is higher;
(3) the biological indicator virus and the indicator cell line contained in the invention exist independently and do not interfere with each other, the virus state is kept stable, the product stably exists in the quality guarantee period and the transportation process, and the detection process is more convenient and faster.
Drawings
FIG. 1 is a graph showing the effect of cobalt source on inactivation of biological indicators at low temperatures (-78 ℃);
FIG. 2 is a graph showing the effect of an accelerator on inactivation of a biological indicator virus at room temperature.
Detailed Description
The technical solutions in the embodiments of the present invention will be 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 protection scope of the present invention.
Referring to fig. 1-2, the present invention provides an embodiment:
example 1: a method for preparing a biological indicator for detecting disinfection and sterilization,
the method comprises the following steps:
s1, taking T-75 cell culture bottles to culture 293T cells, and preparing transfection when the cells grow to 70-80%.
S2, adding 50 mu L Lipo2000 into 1.5ml empty DMEM medium, adding 20 mu g of skeleton plasmid pSG 3-delta env and 5 mu g of envelope plasmid pcDNA3.1-env into 1.5ml DMEM medium, fully mixing, and standing for 5 min.
S3, slowly adding the medium containing the transfection reagent into the medium containing the plasmids, mixing, incubating at room temperature for 20min, and adding into a cell culture flask containing 10mL of empty DMEM medium. Placing the cell culture flask in CO2After 4h incubation in the incubator, 10mL DMEM complete medium (containing 10% FBS) was replaced and incubation was continued for 48 h.
And S4, after 48 hours, collecting virus liquid, centrifuging at 1000rpm for 5min, collecting virus supernatant, subpackaging by 1mL per branch, and storing at-80 ℃ for later use.
Example 2: the biological indicator prepared by the method of example 1, which is applied as a sterilization indicating biological indicator in ozone, ultraviolet rays, cobalt source, accelerator sterilization work.
When applied, the following conditions should be satisfied:
the conditions of ozone disinfection and sterilization are as follows: when the space humidity is more than 75%, the Concentration Time (CT) value is defined as the ozone gas concentration (ppm) multiplied by the administration duration (min), such as 0.05ppm for 20 hours or 0.1ppm for 10 hours.
The conditions of ultraviolet disinfection and sterilization are as follows: is 20-30cm below the radiation source, and has radiation intensity of 0.1mW/cm2Duration 30 seconds, 60 seconds, 300 seconds.
The conditions of cobalt source disinfection and sterilization are as follows: at a temperature of-78 ℃ to 10 ℃ using60Co gamma rays are irradiated by adopting the average dose rate of 0.4kGy/4.5h-3kGy/4.5h, and the total irradiation dose is 2kGy-13 kGy.
The conditions for sterilizing the accelerator are as follows: the biological indicator virus is wrapped on a cell plate and dried in air, and after accelerator irradiation is carried out at room temperature (2-10 ℃), the adsorbed virus is liberated by using a complete culture medium and then detected.
Example 3: the biological indicator for detecting disinfection and sterilization also comprises an indicating cell line used corresponding to the biological indicator, and the indicating cell line is a susceptible cell line of the biological indicator. One of the biological indicator and the indicator cell line carries a luciferase reporter gene.
Example 4: biological indicator for detecting disinfection and sterilization, the biological indicator has an infectious titer TCID50 of 10-4-10-5Per mL, including 10-4/mL、10-5mL and 0.5X10-4/mL。
Example 5: a method for detecting the activity of a biological indicator for sterilization indication, characterized by comprising the steps of:
(1) to the indicated cell dilutions DEAE-Dextran was added at a final concentration of 15. mu.g/mL and the cells were treated at 1X 104The amount per well was added to a 96 well plate.
(2) The biological indicator virus was serially diluted 5-fold.
(3) Detection using a luciferase substrate.
(4) For the calculation of TCID50, the Cutoff value was first calculated, and the Cutoff value was 3xC calr (cell control mean), and the number of positive and negative results were found from the fluorescence values, and TCID50/ml was obtained by Reed-Muench bikes method.
Meanwhile, in order to explore the inactivation effect of different sterilization modes on the biological indicator, the following two comparative examples are provided.
Comparative example 1: the conditions of cobalt source disinfection and sterilization are as follows: at a temperature of-78 ℃ to 10 ℃ using60Co gamma rays are irradiated by adopting the average dose rate of 0.4kGy/4.5h-3kGy/4.5h, and the total irradiation dose is 2kGy-13 kGy. The results are shown in FIG. 1.
Comparative example 2: the conditions for sterilizing the accelerator are as follows: the biological indicator virus is wrapped on a cell plate and dried in air, and after accelerator irradiation is carried out at room temperature (2-10 ℃), the adsorbed virus is liberated by using a complete culture medium and then detected. The results are shown in FIG. 2.
We can see from fig. 1 and 2 that: the two sterilization modes have good inactivation effect on the biological indicator. Is beneficial to the use of the biological indicator as an indicator.
To summarize: the biological indicator is a pseudovirion obtained by modifying and deleting a gene sequence of an envelope protein coding in a euvirus and co-transfecting a 293T cell line with envelope glycoprotein genes of other viruses. One of the biological indicator and the indicator cell line carries a luciferase reporter gene. After the biological indicator virus is used for infecting the indicating cell line, luciferase gene expression is up-regulated, and then the infection and replication efficiency of the biological indicator virus is judged by detecting the expression condition of luciferase. The biological indicator virus based on the luciferase has the advantages of simple preparation, reliable result and the like, and is convenient and rapid to use, high in safety, strong in operability and high in sensitivity.
In conclusion, the beneficial effects of the invention are as follows: (1) the biological indicator virus of the invention is combined with the prior ozone and ultraviolet sterilization biological indicator, namely bacillus subtilis and60compared with a Co irradiation sterilization biological indicator, namely bacillus pumilus, the Co irradiation sterilization biological indicator has higher similarity and higher safety with real viruses; (2) the biological indicator virus detects the expression condition of luciferase by an enzyme-labeling instrument, so that the result is more accurate and the sensitivity is higher; (3) the biological indicator virus and the indicator cell line contained in the invention exist independently and do not interfere with each other, the virus state is kept stable, the product stably exists in the quality guarantee period and the transportation process, and the detection process is more convenient and faster.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (7)
1. The preparation method of the biological indicator for detecting disinfection and sterilization is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing transfection when 293T cells grow to 70-80%, wherein the mass ratio of the skeleton plasmid pSG 3-delta env to the envelope plasmid pcDNA3.1-env is 4: 1;
s2, after transfection for 4h, replacing DMEM complete culture medium (containing 10% FBS), continuing culturing for 48h, and then detoxifying to complete preparation of the biological indicator.
2. A biological indicator for detecting sterilization prepared by the method of claim 1, wherein: the biological indicator used as sterilization indicator is applied to the sterilization work of ozone, ultraviolet rays, cobalt sources and accelerators.
3. A biological indicator for detecting sterilization according to claim 2, wherein: when applied, the following conditions should be satisfied:
the conditions of ozone disinfection and sterilization are as follows: when the space humidity is more than 75%, the Concentration Time (CT) value is defined as the ozone gas concentration (ppm) multiplied by the administration duration (min), such as 0.05ppm for 20 hours or 0.1ppm for 10 hours;
the conditions of ultraviolet disinfection and sterilization are as follows: is 20-30cm below the radiation source, and has radiation intensity of 0.1mW/cm2Duration 30 seconds, 60 seconds, 300 seconds; the conditions of cobalt source disinfection and sterilization are as follows: at a temperature of-78 ℃ to 10 ℃ using60Co gamma rays are irradiated by adopting the average dose rate of 0.4kGy/4.5h-3kGy/4.5h, and the total irradiation dose is 2kGy-13 kGy;
the conditions for sterilizing the accelerator are as follows: the biological indicator virus is wrapped on a cell plate and dried in air, and after accelerator irradiation is carried out at room temperature (2-10 ℃), the adsorbed virus is liberated by using a complete culture medium and then detected.
4. A biological indicator for detecting sterilization according to claim 2 or 3, wherein: the biological indicator has an infectious titer of TCID50 of 10-4-10-5/mL。
5. A biological indicator for detecting sterilization according to claim 4, wherein: also comprises an indicating cell line used corresponding to the biological indicator, and the indicating cell line is a susceptible cell line of the biological indicator.
6. A biological indicator for detecting sterilization according to claim 5, wherein: one of the biological indicator and the indicator cell line carries a luciferase reporter gene.
7. A method for detecting the activity of a biological indicator for sterilization indication according to claim 5 or 6, characterized by comprising the steps of:
(1) to the indicated cell dilutions DEAE-Dextran was added at a final concentration of 15. mu.g/mL and the cells were treated at 1X 104The amount per well was added to a 96 well plate;
(2) sequentially diluting the biological indicator viruses by 5 times;
(3) detection using a luciferase substrate;
(4) for the calculation of TCID50, the Cutoff value was first calculated, and the Cutoff value was 3xC calr (cell control mean), and the number of positive and negative results were found from the fluorescence values, and TCID50/ml was obtained by Reed-Muench bikes method.
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| CN114480558A (en) * | 2020-10-23 | 2022-05-13 | 亚丁卫生耳鼻喉科 | Biological indicator for rapid verification of disinfection or sterilization |
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