CN114134140B - A sputum preservation lysis solution and its kit and application - Google Patents
A sputum preservation lysis solution and its kit and application Download PDFInfo
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Abstract
The invention provides a sputum preservation lysate, a kit and application thereof, wherein the sputum preservation lysate comprises the following components: 0.5-1.5M strong lye selected from: any one of NaOH and KOH; 5-12% of a surfactant selected from the group consisting of: at least one of Triton X-100, SDS, N-lauroyl sarcosine sodium, tween-80, CTAB or a combination thereof; 4-8M protein denaturant selected from: any one or combination of guanidine hydrochloride, TCEP hydrochloride and urea; 0.05-0.15M metal ion chelating agent; a proper amount of preservative; and 12-20% v/v wax-dissolving agent selected from: petroleum ether, pentane, hexane, chloroform, or a combination thereof. The sputum preservation lysate integrates multiple functions of sample preservation, inactivation, liquefaction, wall breaking, cracking, protein sedimentation and the like, is simple and convenient to operate, has reliable results, and provides a new idea for rapid diagnosis of multiple pathogens.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a sputum preservation lysate, a kit and application thereof.
Background
Detection of most respiratory pathogens requires extraction of their nucleic acids. Different extraction methods and procedures need to be established for different sample types and pathogen characteristics. Among these pathogens, extraction of Mycobacterium tuberculosis has been one of the clinical challenges.
Mycobacterium tuberculosis (Mycobacterium tuberculosis), commonly known as tubercle bacillus or tubercle bacillus, is a kind of bacteria with obligate aerobic property, and has positive acid staining resistance. The bacillus is pathogenic bacteria causing tuberculosis of human and animals, can invade all organs of the whole body, but mainly takes tuberculosis as the most common cause of oxygenation, and the tuberculosis is an ancient disease, takes a global distribution situation and occupies the first cause of death in bacterial infectious diseases. Therefore, the method has the advantages of timely, efficient and accurate detection of the mycobacterium tuberculosis, and plays an important role in preventing and treating diseases caused by the mycobacterium tuberculosis.
However, the mycobacterium tuberculosis requires 4-5 weeks of in vitro culture whether it is an acid-fast staining method of Ziehl-Neelsen or other traditional diagnostic methods, and the method is long in time, complex in operation, easy to pollute, and easy to cause false negative results and a large number of missed detection. The molecular biological diagnosis technology can obtain positive results by only containing a few bacteria in each milliliter, has simple operation, high specificity and shorter time consumption, and can rapidly diagnose the mycobacterium tuberculosis, thereby being rather favored by clinical hospital tuberculosis diagnosis. However, this technique requires a certain degree of purity and concentration of DNA in the sample, and the cell wall of Mycobacterium tuberculosis contains a large amount of lipid, so that the common lysozyme, protease and even lysing agent cannot completely rupture the cell membrane of Mycobacterium tuberculosis to release nucleic acid.
There are domestic literature reports on a method for preserving sputum and a method for extracting DNA of Mycobacterium tuberculosis in sputum, the method comprising: (1) sputum liquefaction: transfer sputum containing mycobacterium tuberculosis to TE buffer after liquefaction with 1N NaOH, (2) inactivate in combination with mycobacterium: under the condition of 75-85 ℃, the bacteria are inactivated by 30-60 minutes of treatment, (3) the bacteria are broken and cracked: then the purpose of bacterial disruption is achieved through vortex oscillation and ultrasonic dispersion treatment, then lysozyme is added and water bath digestion is carried out for 2-24 hours at 37 ℃ to obtain a wall breaking product, the time is long, the operation is complex, and the laboratory pollution and the probability of operator infection are increased more easily.
Obviously, the existing methods for liquefying and inactivating sputum preservation fluid and extracting nucleic acid have the following defects:
1) The inactivation takes a long time. Different from common bacteria which are inactivated at high temperature for 5-10 minutes, the Mycobacterium tuberculosis has very strong resistance to the action of physical and chemical factors due to the fact that the waxy structure in the cell wall accounts for about 60 percent, and the Mycobacterium tuberculosis needs to be inactivated by adopting a method of treating for 30-60 minutes at 75-85 ℃.
2) The sample needs to be liquefied. Because of the specificity of the sputum sample, the sample needs to be liquefied by adding 1N NaOH, then transferred to a preservation solution at 75-85 ℃ for 30-60 minutes for inactivation, and then is oscillated for liquefaction, and a special reagent is added into some more complex nucleic acid extraction reagents for oscillation liquefaction.
3) Cracking is difficult. The common lysozyme or protease can not completely rupture cell membranes to release nucleic acid, so that PCR false negative causes detection omission, and the wall breaking and inactivation time is long and the operation is complicated, thus being not suitable for clinical large-scale popularization and application.
Therefore, establishing a sputum preservation and lysis solution suitable for detecting the tubercle bacillus, which is used for solving the problems of long time consumption, complex operation, easy pollution, missed detection caused by high false negative and incapability of having the functions of preserving solution and nucleic acid extraction and lysis solution in the existing respiratory pathogens detection method, especially for detecting the tubercle bacillus, becomes one of the main problems to be solved urgently in clinic.
Disclosure of Invention
The invention aims to provide a sputum preservation lysate, a kit and application thereof, so as to solve the problems that in the prior art, a tubercle bacillus detection method is long in time consumption, complex in operation, easy to pollute, high in false negative and cause detection omission, and the existing sputum preservation lysate cannot have the functions of preserving the lysate and extracting nucleic acid.
In order to solve the technical problems, the invention adopts the following technical scheme:
According to a first aspect of the present invention there is provided a sputum preserving lysate comprising: 0.5-1.5M strong alkali solution, wherein the strong alkali solution is selected from the group consisting of: any one of NaOH and KOH; 5-12% (g/100 mL) of a surfactant selected from the group consisting of: at least one of Triton X-100, SDS, N-lauroyl sarcosine sodium, tween-80, CTAB or a combination thereof; 4-8M protein denaturant selected from the group consisting of: any one or combination of guanidine hydrochloride, TCEP hydrochloride and urea; 0.05-0.15M metal ion chelating agent; a proper amount of preservative; and 12-20% v/v wax-dissolving agent selected from the group consisting of: petroleum ether, pentane, hexane, chloroform, or a combination thereof.
The content of the wax dissolving agent is 16-18%v/v.
The preservative is selected from the group consisting of: any one or combination of streptomycin sulfate, potassium benzoate and potassium sorbate.
The metal ion chelating agent includes, but is not limited to, EDTA.
The content of the preservative is 0.2-0.8% (g/100 mL).
According to a preferred scheme of the invention, a sputum preservation lysate is provided, which comprises the following components in percentage by weight: KOH 0.8-1.2M, guanidine hydrochloride 4-8M, TCEP hydrochloride 1-3%, triton X-100-3%, N-lauroyl sarcosinate 3-4%, tween-80-2%, EDTA 0.05-0.15M, CTAB 1-3%, petroleum ether 14-18%, streptomycin sulfate 40-60 mg/L, and potassium benzoate 0.4-0.6%.
According to another preferred scheme of the invention, a sputum preservation lysate is provided, which comprises the following components in percentage by weight: naOH 0.8-1.2M, guanidine hydrochloride 4-8M, TCEP hydrochloride 1-3%, triton X-100-4%, N-lauroyl sarcosinate 3-5%, EDTA 0.05-0.15M, CTAB 1-3%, petroleum ether 14-18%, streptomycin sulfate 40-60 mg/L, potassium sorbate 0.4-0.6%.
According to a further preferred scheme of the invention, a sputum preservation lysate is provided, which comprises the following components in percentage by weight: 1.0 to 1.2M of NaOH, 6 to 8M of guanidine hydrochloride, 2 to 3 percent of TCEP hydrochloride, 2 to 3 percent of Triton X-100, 3 to 4 percent of N-lauroyl sarcosinate, 0.1 to 0.15M of EDTA, 2 to 3 percent of CTAB, 16 to 18 percent of petroleum ether, 50 to 60mg/L of streptomycin sulfate and 0.5 to 0.6 percent of potassium sorbate.
According to a further preferred scheme of the invention, a sputum preservation lysate is provided, which comprises the following components in percentage by weight: naOH 1M, guanidine hydrochloride 6M, TCEP hydrochloride 2%, triton X-100, N-lauroyl sarcosine sodium 4%, EDTA 0.1M, CTAB 2%, petroleum ether 16%, streptomycin sulfate 50mg/L, potassium sorbate 0.5%.
It should be understood that in the present specification, "%" of other components means g/100mL except that "%" of wax-dissolving agent is v/v.
According to a second aspect of the present invention there is provided a kit comprising a sputum preserving lysate as described above.
According to a third aspect of the present invention, there is provided an application of the sputum preserving lysate described above, the application comprising: the sputum preservation and lysis, oropharynx swab, nasopharyngeal swab, hydrothorax, alveolar lavage and other types of samples are preserved and lysed, and the method is applicable to detection of other respiratory pathogens besides detection of tubercle bacillus.
In order to overcome the defects that in the prior art, the inactivation time is long, the dissolving speed of preservation solution on sputum is low, the wall breaking step is complex and the time is long, the lysate needs to be added or replaced in the extraction process, and the tubercle bacillus in the sputum cannot be rapidly and effectively extracted, the preservation lysate capable of rapidly lysing the tubercle bacillus is provided, and the preservation lysate capable of rapidly lysing the tubercle bacillus can be used as sample preservation solution by adding the tubercle sputum preservation lysate into a sample at one time, and can be directly used as the lysate for nucleic acid extraction without replacing or adding other components after entering a laboratory to directly carry out subsequent nucleic acid extraction, so that the complex and complicated operations of inactivating, liquefying and breaking the wall are saved.
The key invention is that an oily wax-dissolving agent such as petroleum ether or a substitute thereof is added into the preservation lysate for the first time, so that 60% of waxy components contained in the cell wall of the mycobacterium tuberculosis are dissolved, and the problem of long time consumption in inactivation in the prior art is solved. However, none of the sputum lysates of the prior art employ such a component.
The other key invention point is that by providing a combined preservation lysate component comprising strong alkali, surfactant, protein denaturant, metal ion chelating agent, wax-dissolving agent and preservative, the full lysis of mycobacterium tuberculosis and the complete denaturation of protein can be realized, the reliability and the accuracy of detection are ensured, and meanwhile, the preservation lysate component also has the preservation function. The sputum treated by the preservation lysate has no obvious difference between the sputum extraction effect and the detection result stored for 7 days, 30 days, 90 days, 120 days and 360 days compared with the sputum stored for 0 day.
Sample types for which the subject kits are useful are demonstrated by subsequent clinical sample verification include, but are not limited to, sputum, oropharyngeal swab, nasopharyngeal swab, hydrothorax, and alveolar lavage. Pathogens involved in the detection include, but are not limited to, mycobacterium tuberculosis, influenza a virus, influenza b virus, adenovirus, metapneumovirus.
In summary, according to the sputum preservation lysate provided by the invention, the functions of sample preservation, inactivation, liquefaction, wall breaking and cracking, protein sedimentation, removal of surface saccharide impurities and the like are integrated, so that the sputum preservation lysate can be used for preserving and cracking sputum, preserving samples of the type such as oropharynx swab, nasopharyngeal swab, hydrothorax and alveolar lavage liquid, detecting other respiratory pathogens, greatly reducing the probability of laboratory pollution and pathogen infection of operators, and is simple and convenient to operate, reliable in result, and capable of greatly saving nucleic acid extraction time, and providing a new idea for rapid diagnosis of mycobacterium tuberculosis and other multiple pathogens.
Drawings
FIG. 1 shows the results of Ct values obtained by adding fresh sputum to the preserved lysate and leaving the same at 37℃for 0, 7, 30, 90, 120, 360 days followed by nucleic acid extraction and fluorescent quantitative PCR detection;
FIG. 2 shows the results of nucleic acid extraction and airway multiplex RT-qPCR detection using the preserved lysate for oropharyngeal swabs; wherein, the influenza A is 1-B, 2-adenovirus, 3-metapneumovirus and 4-influenza A;
FIG. 3 shows the results of nucleic acid extraction and airway multiplex RT-qPCR detection using the preserved lysate for nasopharyngeal swab; wherein, the influenza A is 1-B, 2-adenovirus, 3-metapneumovirus and 4-influenza A;
FIG. 4 shows the results of nucleic acid extraction and airway multiplex RT-qPCR detection using the preserved lysate for hydrothorax; wherein, the influenza A is 1-B, 2-adenovirus, 3-metapneumovirus and 4-influenza A;
FIG. 5 shows the results of nucleic acid extraction of alveolar lavage fluid from the stored lysate and performing multiple RT-qPCR assays of respiratory tract; wherein, the influenza A is 1-B, 2-adenovirus, 3-metapneumovirus and 4-influenza A.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
According to this embodiment, there is provided a preservation lysate comprising: naOH 0.5M, guanidine hydrochloride 1M, CTAB 4%, urea 3M, DTT 2%, triton X-100%, EDTA 0.1M.
The preserved lysate of the formula is used for treating sputum, and the result shows that: A260/A280 ratio is low, there is an effect of protein, nucleic acid concentration is low (measured 30% lower than the original concentration), and cleavage may be insufficient. It is suggested to increase the components of cleavage and protein denaturation.
Example 2
According to this embodiment, there is provided a preservation lysate comprising: naOH 0.5M, guanidine hydrochloride 2M, urea 3M, triton X-100%, EDTA 0.1M, N-lauroyl sarcosine sodium 4%, tween 80 2% and SDS 2%.
The preserved lysate of the formula is used for treating sputum, and the result shows that: the lower concentration of nucleic acid (measured 20% lower than the original concentration) may be due to insufficient lysis of Mycobacterium tuberculosis. It is suggested to further increase the components contributing to the lysis of the cells.
Example 3
According to this embodiment, there is provided a preservation lysate comprising: naOH 0.5M, guanidine hydrochloride 4M, TCEP hydrochloride 2%, triton X-100%, EDTA 0.1M, N-lauroyl sarcosine sodium 4%, tween 80 2%, SDS 2% and petroleum ether 16%.
The preserved lysate of the formula is used for treating sputum, and the result shows that: and (3) during the sample loading and cracking step, continuously overflowing the cracking liquid when the sample magnetic rod sleeve is stirred and mixed, and prompting that the content of the surfactant is too high. It is suggested to reduce the amount of surfactant.
Example 4
According to this embodiment, there is provided a preservation lysate comprising: naOH 1M, guanidine hydrochloride 6M, TCEP hydrochloride 2%, triton X-100%, EDTA 0.1M, CTAB 2%, N-lauroyl sarcosine sodium 4%, petroleum ether 16%, streptomycin sulfate 50mg/L.
The preserved lysate of the formula is used for treating sputum, and the result shows that: the formula is used as a pyrolysis liquid to verify qualification, and then preservative components of a preservation liquid are added to enable the system to achieve the preservative preservation capacity.
Example 5
According to this embodiment, there is provided a preservation lysate comprising: naOH 1M, guanidine hydrochloride 6M, TCEP hydrochloride 2%, triton X-100, EDTA 0.1M, CTAB 2%, N-lauroyl sarcosine sodium 4%, petroleum ether 16%, streptomycin sulfate 50mg/L, and potassium benzoate 0.5%.
The preserved lysate of the formula is used for treating sputum, and the result shows that: after the preservative component potassium benzoate is added, the result is good, and the potassium sorbate can be better judged by replacing the potassium sorbate as another preservative.
Example 6
According to this embodiment, there is provided a preservation lysate comprising: naOH 1M, guanidine hydrochloride 6M, TCEP hydrochloride 2%, triton X-100, EDTA 0.1M, CTAB 2%, N-lauroyl sarcosine sodium 4%, petroleum ether 16%, streptomycin sulfate 50mg/L, potassium sorbate 0.5%.
The preserved lysate of the formulation was used for sputum treatment, and the results are shown in Table 1 below, with better results with the addition of the preservative potassium sorbate, and the formulation was selected as the final formulation of the preserved lysate of tuberculosis sputum.
TABLE 1
Fresh sputum of strong positive, medium positive and weak positive of the mycobacterium tuberculosis is added into the preservation lysate and is preserved for 7 days, 30 days, 90 days and 120 days at 37 ℃ for nucleic acid extraction and self-built fluorescent quantitative PCR of the mycobacterium tuberculosis is carried out for 1081 and 6110 detection of the tuberculosis complex, and the preservation time is judged by observing the sample positive coincidence rate and Ct value change with 0 day. The results are shown in FIG. 1. Wherein, tuberculosis 1081 and tuberculosis 6110 refer to IS1081 gene and IS6110 gene of Mycobacterium tuberculosis, respectively.
Analysis of results: the sputum extraction effect and the detection result of the system stored for 7 days, 30 days, 90 days, 120 days and 360 days are not significantly different from those of the sputum extraction effect and the detection result stored for 0 day.
Example 7
According to this embodiment, there is provided a preservation lysate comprising: naOH 1M, guanidine hydrochloride 6M, TCEP hydrochloride 2%, triton X-100, EDTA 0.1M, CTAB 2%, N-lauroyl sarcosine sodium 4%, petroleum ether 16%, streptomycin sulfate 50mg/L, potassium sorbate 0.5%.
The lysate is used for nucleic acid extraction of samples of the type including influenza a, influenza b, adenovirus, metapneumovirus, oropharynx swab, nasopharyngeal swab, pleural effusion and alveolar lavage (as upper set of curves in fig. 2-5), and simultaneously the samples are subjected to 250-fold dilution and parallel control (as lower set of curves in fig. 2-5) for extraction and respiratory multiplex RT-qPCR detection of the extracted nucleic acids. The detection results are shown in table 2 and fig. 2 to 5.
TABLE 2
Analysis of results: the system is used for multiple detection of influenza A, influenza B, adenovirus and metapneumovirus by oropharynx swab, nasopharyngeal swab, hydrothorax and alveolar lavage liquid, has no obvious influence on detection results, and shows that the preservation and pyrolysis liquid is also suitable for preservation and pyrolysis of oropharynx swab, nasopharyngeal swab, hydrothorax and alveolar lavage liquid type samples.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
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| CN107904232A (en) * | 2017-12-29 | 2018-04-13 | 浙江今复康生物科技有限公司 | A kind of method of the rapid extraction nucleic acid from sputum |
| CN108823282A (en) * | 2017-04-28 | 2018-11-16 | 利多(香港)有限公司 | Sample nucleic acid detection kit, reagent and application thereof |
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| US9683256B2 (en) * | 2007-10-01 | 2017-06-20 | Longhorn Vaccines And Diagnostics, Llc | Biological specimen collection and transport system |
| EP2535428B1 (en) * | 2007-10-01 | 2015-09-09 | Longhorn Vaccines and Diagnostics, LLC | Biological specimen collection and transport system and methods of use |
| AU2015268067A1 (en) * | 2014-05-27 | 2016-12-22 | Dna Genotek Inc. | Composition and method for stabilizing and maintaining the viability of hardy microorganisms |
| EP3501282A1 (en) * | 2017-12-22 | 2019-06-26 | F. Hoffmann-La Roche AG | Composition for processing a sputum sample |
| CN112680547B (en) * | 2020-03-13 | 2023-07-07 | 苏州白垩纪生物科技有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit and application thereof |
| CN111218444B (en) * | 2020-04-24 | 2020-09-01 | 广州安必平医药科技股份有限公司 | Sputum preserving fluid |
| CN111926059A (en) * | 2020-08-23 | 2020-11-13 | 广州源古纪科技有限公司 | Novel preservation solution for microbial nucleic acid in sputum |
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| CN108823282A (en) * | 2017-04-28 | 2018-11-16 | 利多(香港)有限公司 | Sample nucleic acid detection kit, reagent and application thereof |
| CN107904232A (en) * | 2017-12-29 | 2018-04-13 | 浙江今复康生物科技有限公司 | A kind of method of the rapid extraction nucleic acid from sputum |
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