CN108251496B - Microorganism mass spectrum detection capability verification article and preparation method thereof - Google Patents
Microorganism mass spectrum detection capability verification article and preparation method thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6872—Methods for sequencing involving mass spectrometry
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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Abstract
The invention relates to a microorganism mass spectrum inspection capability verification article and a preparation method thereof, wherein the microorganism mass spectrum inspection capability verification article comprises a capability verification article basic preparation and a capability verification article additive, namely a pure culture inactivated bacteria liquid/bacterial colony of a target detection strain in a current capability verification plan or a pure culture inactivated bacteria preparation of the target detection strain after vacuum freeze drying, and the capability verification article is mixed with the capability verification article additive to obtain the microorganism mass spectrum inspection capability verification article. The invention overcomes the defects that the existing capacity verification article adopts viable bacteria preparation, has biological potential safety hazard, is easy to be repeatedly detected by a plurality of detection methods at the same time, has long return result period and the like, is safe and quick, is suitable for a microorganism mass spectrum inspection capacity verification plan, and better realizes the expected purpose of the capacity verification plan.
Description
Technical Field
The invention belongs to the field of microbial detection, and particularly relates to a microbial mass spectrometry detection capability verification article and a preparation method thereof.
Background
In recent years, with the development of scientific technology, on the basis of the traditional clinical microorganism detection method, new detection technologies are gradually developed, particularly, a microorganism rapid identification technology and a microorganism rapid identification method appear, the diagnosis speed of clinical infectious diseases is improved, and the method has important clinical application value and social significance for reasonably using antibacterial drugs and timely rescuing the lives of patients. Among them, the microbial mass spectrometry technology has been mature and gradually applied to clinical microbial testing.
In order to ensure the successful popularization of the inspection quality and clinical application of the new inspection technology, the health administration department, especially the clinical inspection quality supervision and management department, needs to research and establish new quality supervision methods and technical standards in time, strengthen the quality supervision and management of new instruments, new equipment and new technology, and ensure the safety and high efficiency of clinical diagnosis and treatment work.
Mass spectrometry was originally used for industrial microbiological testing. At present, mass spectrometry is officially approved in many countries, including China, for clinical microbiological examination, mainly for rapid identification of pathogenic bacteria and fungi. Currently, capacity verification providers who formally develop a capacity verification plan for mass spectrometry testing of microorganisms internationally have few reports. In China, a plurality of provincial and municipal health care institutions and scientific research institutions in China have successively developed a pathogenic microorganism mass spectrometry inspection technology which is directly or indirectly used for diagnosing clinical infectious diseases. At present, in an organization approved by China qualification national Committee (CNAS) and capable of providing a verification plan (PTP) for the capability of clinical microorganism inspection, a verification plan for the capability of mass spectrometry of microorganisms is formally developed by the clinical inspection center in Shanghai city. The existing capacity verification article for the microbial mass spectrometry provided by the mechanism is basically the same as the capacity verification article of the conventional bacteria identification/drug sensitivity test method for evaluating an automatic microorganism identification instrument/drug sensitivity test system, namely, a certain amount of bacterial colony/bacterial liquid is added into skim milk to prepare bacterial suspension, and the bacterial suspension is subjected to vacuum freeze drying and then is used as the capacity verification article to be distributed to a microbial laboratory. After the latter is cultured, the target bacteria are selected and identified by mass spectrum technology. The product is used for verifying the capability of the microorganism mass spectrum inspection, and has the following defects:
first, the capacity-verifying item remains a live bacterial preparation, presenting a certain biosafety risk. In particular, some highly pathogenic/infectious pathogenic bacteria live bacteria cannot be distributed to a laboratory as a capability verification article, the identification capability of the clinical laboratory on the bacteria cannot be exercised and verified, and the bacteria may be encountered in clinical work, so that it is difficult to ensure that the laboratory can quickly and correctly identify the bacteria, and even cause the infection in the laboratory. For example, many clinical laboratory test personnel nationwide have developed brucella laboratory infections in recent years;
secondly, the microorganism mass spectrometry capability verification articles are easy to be distinguished and repeatedly detected by a multi-detection system, and the expected purpose of the capability verification plan cannot be achieved. The purpose of the capacity verification plan is to evaluate the comprehensive capacity of bacteria identified by a laboratory through a mass spectrometry technology and the working state of an instrument. However, in order to achieve good results, some laboratories may adopt an automated microorganism identification instrument and a mass spectrometry technology to detect the capability verification articles at the same time, and individual laboratories even adopt a molecular biology technology at the same time to mutually verify, select the best identification result to return, and cannot truly reflect the microorganism mass spectrometry detection capability;
thirdly, the time for returning the identification result is delayed obviously, the rapid diagnosis advantage of the mass spectrometry detection technology cannot be embodied, and the risk of data collusion among laboratories is increased. The mass spectrometry technology for identifying bacteria is simple and convenient to operate, and an identification result can be obtained within a few minutes, which is an obvious advantage of the technology and also a main reason for introducing the technology in a laboratory. The current capacity verification article needs to start from bacterial culture, separation and purification, generally needs about one week to formally return an identification result, is basically consistent with the result return time of an automatic microorganism identification instrument, and is not suitable for flight inspection activities of laboratory quality assessment. Meanwhile, the reporting time of the result is prolonged, and the risk of data collusion among laboratories can also be increased.
Disclosure of Invention
The invention aims to solve the technical problem of providing a microorganism mass spectrometry test capability verification article and a preparation method thereof, the capability verification article overcomes the defects that the existing capability verification article adopts viable bacteria preparation, has biological potential safety hazard, is easy to be repeatedly detected by a plurality of detection methods at the same time, has long return result period and the like, is safe and quick, is suitable for a microorganism mass spectrometry test capability verification plan, and can better realize the expected purpose of the capability verification plan.
The invention provides a microorganism mass spectrum inspection capability verification article, which comprises a capability verification article basic preparation, namely a pure culture inactivated bacteria liquid/bacterial colony of a target detection strain in a current capability verification plan, or a pure culture inactivated bacteria liquid preparation of the target detection strain after vacuum freeze drying.
The diluent of the pure culture inactivated bacterial liquid/bacterial colony of the target detection strain is sterile distilled water, deionized water or a liquid culture medium, or 10-100g/L bovine serum albumin solution is continuously added into the diluent.
When the additive of the capacity verification article is not added, the invention is used for indoor quality control of the microbial mass spectrometry inspection technology.
The invention also includes the additive of the capacity verification article, namely one or more purified genome DNA fragments of bacteria or fungi of different species or different species from the target detection strain in the known current capacity verification plan or a mixture of the purified genome DNA fragments thereof. The function of the additive of the capability-verifying article is to enable the capability-verifying article to meet the requirement of conventional PCR amplification without interfering with the direct identification of bacteria/fungi by adopting a mass spectrometry technology.
The method for obtaining the additive of the capability verification article comprises the following steps:
designing 2 pairs of amplified bacteria 16S rDNA primers, wherein 1 pair is an amplified bacteria 16S rDNA universal primer, and the upstream and downstream of an amplification product of the other 1 pair of primers cover the sequence of a bacteria 16S rDNA universal primer binding region;
or designing 2 pairs of primers for amplifying an internal transcribed spacer 2ITS-2 between 5.8S rDNA and 28S rDNA of the fungus, 1 pair of primers for amplifying the ITS-2 of the fungus and the sequence of the universal primer binding region of the ITS-2 gene covering the upstream and downstream of an amplification product of the other 1 pair of primers;
and (3) respectively amplifying by using the designed primers to obtain the primer.
The mixing volume ratio of the basic preparation of the capability verification article to the additive of the capability verification article is 1: 1-10.
The invention also provides a preparation method of the microorganism mass spectrometry detection capability verification article, which comprises the following steps:
(1) taking a pure culture bacterial colony of a target detection strain in the capacity verification plan or a liquid culture centrifugal precipitate thereof, adding absolute ethyl alcohol, and uniformly mixing to inactivate and fix bacteria; centrifuging to remove ethanol, precipitating to room temperature, and volatilizing residual ethanol; adding sterile distilled water to prepare a bacterial liquid for later use;
(2) selecting pure culture of common clinical pathogenic bacteria different from a target detection strain, extracting sufficient purified genome DNA of the pathogenic bacteria, adjusting the concentration to meet the requirement of conventional PCR (150pg/ml) through absorbance measurement, subpackaging, and immediately using or carrying out vacuum freeze drying; designing primer for amplification, regulating the concentration of the obtained amplification product, subpackaging (more than 10 times of the concentration of the template required by conventional PCR), and immediately using or vacuum freeze-drying;
(3) adding the basic preparation of the capability verification article in the step (1) into the additive of the capability verification article in the step (2), and mixing to prepare a liquid preparation of the capability verification article (used on the same day);
or continuously adding bovine serum albumin, freezing the liquid preparation overnight, and freeze-drying under vacuum to obtain lyophilized preparation of the capacity verification article, and storing at-40 deg.C.
The clinically common pathogenic bacteria in the step (2) comprise one or more of aerobic bacteria (gram-positive bacteria and gram-negative bacteria), anaerobic bacteria and fungi (candida and filamentous fungi).
Based on the characteristics of the microbial mass spectrometry technology, the invention designs a capacity verification article for a microbial mass spectrometry capacity verification plan and discloses a preparation method of the capacity verification article. The invention overcomes the defects that the existing capacity verification article adopts viable bacteria preparation, has biological potential safety hazard, is easy to be simultaneously and repeatedly detected by a plurality of detection methods, has long return result period and the like, is safe and quick, is only suitable for a microorganism mass spectrum inspection capacity verification plan, and can better realize the expected purpose of the capacity verification plan. The invention can be used for capacity verification plans, can also be used for commercial purposes such as indoor quality control products of a microorganism identification mass spectrometer MALDI-TOF, instrument performance evaluation and the like, and has the advantages of safety, science and wide market application prospect.
The invention aims to provide a forming bracket (in a block shape) for bacterial liquid in the vacuum freeze drying process and generate certain adhesion to the wall of a test tube so as not to fly away in a powder shape during vacuum pumping. The classical method is adding milk, but the milk contains a plurality of proteins, sugar, polypeptide and other components which are complex, and the molecular weight of some macromolecular substances can be close to the molecular weight of specific protein when identifying bacteria by mass spectrum technology, so that the identification is interfered, and the method is not suitable.
Advantageous effects
(1) The invention is based on the characteristic that the microorganism mass spectrum inspection technology does not need to identify the survival of the strain, adopts the inactivated strain preparation, and not only overcomes the potential biological safety hazard existing in the prior capacity verification article adopting the live strain preparation; moreover, as the target tested strain is an inactivated preparation, the possibility that a laboratory adopts an automatic microorganism identification instrument based on a bacterial culture method to carry out repeated identification comparison of a multi-identification system is also avoided; the addition of bovine serum albumin with known molecular weight (molecular weight is about 66.446kDa) instead of skimmed milk with complex components not only helps to shape the bacterial mass during freeze drying, but also does not interfere with the normal identification of bacteria by mass spectrometry (bacterial proteins with molecular weight in the region of 2-20kDa have the most characteristic of mass spectrometry).
(2) The invention also comprises a DNA additive which can effectively interfere the repeated identification of the target strain to be detected by directly adopting a molecular biology method based on the principle of DNA amplification/sequencing in a laboratory, so that the invention is only suitable for identifying the target strain to be detected by adopting a mass spectrometry technology and is beneficial to realizing the capacity verification plan target;
(3) the capacity verification article contains a proper amount of target bacteria to be detected, can be directly used for mass spectrum identification, does not need enrichment culture, can obtain an identification result within minutes, is not only suitable for a conventional indoor evaluation method of microbial mass spectrum inspection, but also is more suitable for indoor evaluation flight inspection, and the identification result is 'right-waiting' and acceptable; the freeze-dried product is suitable for cold-chain long-distance transportation, can effectively prolong the shelf life, is safer and more scientific, and has wide market application prospect.
Drawings
FIG. 1 is a diagram showing verification of the identification ability of Escherichia coli (score value 2.183) in an article by using BRUKER microorganism identification mass spectrometer (MALDI-TOF);
FIG. 2 shows the successful amplification of the 16S rRNA gene fragment of Escherichia coli from a capacity-verified article using PCR;
FIG. 3 shows the successful amplification of Acinetobacter baumannii 16S rRNA gene fragments from capacity-verified articles using PCR technology;
FIG. 4 shows that 2 16S rRNA gene fragments are amplified by PCR in the capacity verification article, wherein the sequencing result of one 16S rRNA gene amplification fragment is registered in NCBI Blast comparison, and has 99% homology with the 16S rRNA gene fragment of Escherichia coli (Y29R 2); FIG. 5 shows that 2 kinds of 16S rRNA gene fragments are amplified by PCR in the capacity verification article, and the sequence of another 16S rRNA gene amplified fragment is registered with NCBI Blast comparison, and has 100% homology with 16S rRNA gene fragment of Acinetobacter baumannii (AB-01).
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Preparing a basic preparation of the capacity verification article:
the Escherichia coli is taken as an example of a target detection bacterium in a capacity verification plan. Culturing 3-5 (about 20mg) colonies of the strain on a blood plate at 35 deg.C for 18h, adding into a sterile EP tube containing 0.5ml of anhydrous ethanol, mixing, acting at room temperature for 30ml to inactivate and fix bacteria; centrifuging at 4,000r/min to remove ethanol, shaking the precipitate in EP tube for 2s to disperse, standing at room temperature (22-25 deg.C), and volatilizing residual ethanol; adding 0.2ml of sterile distilled water into the precipitate again, mixing uniformly, and subpackaging 0.5ml of sterile sharp-bottomed EP tubes with 40 mu l of each tube for instant use; can also be stored at low temperature of-40 ℃ for 1 week; the preparation method can be used for preparing the product in batches.
Preparation of additive for capacity verification article:
taking acinetobacter baumannii 16S rRNA genome DNA as an additive of a capability verification article as an example: taking Acinetobacter baumannii pure culture centrifugal precipitate (about 20mg), adopting a DNA extraction and purification kit of ROCHE company, operating according to a specification, extracting high-quality purified genome DNA of the Acinetobacter baumannii, subpackaging, and immediately using (or vacuum freeze drying and storing at-80 ℃ low temperature);
TaKaRa Ex Taq is adoptedTMPCR reaction kit, general primer sequence:
F：5ˊ-AGAGTTTGATCMTGGCTCAG-3ˊ，
R：5ˊ-GGTTACCTTGTTACGACTT-3ˊ；
the primers were synthesized by Takara Shuzo (Dalian) Co., Ltd.
Reaction system: mu.l 10 Xbuffer, 4. mu.l MgCl were taken separately2(25mmol/L), 5. mu.l dNTPmix (10. mu. mol/L), 2. mu.l each of primers (10. mu. mol/L), 4. mu.l of DNA template, 2.5U of Taq DNA polymerase, and make up to a total volume of 50. mu.l with sterile ultrapure water.
The reaction conditions are as follows: pre-denaturation at 94 ℃ for 1min, 15s at 98 ℃, 60s at 55 ℃ and 60s at 72 ℃ for 30 cycles in total. After PCR amplification, 3. mu.l of PCR reaction product was subjected to agarose gel electrophoresis to confirm that the PCR amplification was successful. Subpackaging the PCR product with 50 μ l per tube for immediate use or storing at-70 deg.C; can be prepared in batches by the same method.
Preparing a microorganism mass spectrum detection capability verification article:
taking 10 mu l of freshly prepared acinetobacter baumannii 16S rRNA gene universal primer PCR amplification product (or PCR product stored at ultralow temperature of-70 ℃) as a capability verification article additive, adding the capability verification article additive into prepared 40 mu l of capability verification article basic preparation (escherichia coli inactivated bacterial liquid), and mixing to prepare a capability verification article liquid preparation; adding 50 μ L bovine serum albumin with concentration of 100g/L, mixing, freezing the liquid preparation at-40 deg.C overnight, vacuum freeze drying, and making into lyophilized preparation for capacity verification product, and storing at-40 deg.C. Cold chain transfer at 2-8 ℃ requires a laboratory to receive the capability verification article to complete the identification and feed back the result on the same day.
The application of the microorganism mass spectrum inspection technology capability verification article:
and (3) adding 0.2ml of sterilized ultrapure water into the prepared capacity verification article, repeatedly blowing, sucking and uniformly mixing for several times to dissolve the capacity verification article, taking a proper amount of bacteria liquid precipitate on a target spot of a MALDI-TOF target plate, coating the bacteria liquid precipitate into a thin layer, and operating according to a conventional MALDI-TOF detection method.
And verifying the article by adopting PCR amplification and sequencing technology to detect the capability. Taking the prepared capacity verification article, adopting a DNA extraction and purification kit of ROCHE company, and operating according to a specification to extract the genomic DNA of the target bacteria (Escherichia coli) in the capacity verification article; TaKaRa Ex Taq is adoptedTMThe PCR reaction kit comprises the same general primers, reaction systems and reaction conditions; after PCR amplification, 3. mu.l of PCR reaction product was subjected to agarose gel electrophoresis to confirm that the PCR amplification was successful. Sequencing the PCR product by Venezhi Bioengineering (Shanghai) Co., Ltd; meanwhile, connecting the PCR product with a pMD18-T vector to construct a pMD18-T plasmid, transferring the plasmid into competent bacteria to construct clone, and accurately sequencing by a member of the Token biological engineering (Shanghai) corporation; the sequencing result is registered with NCBI for Blast comparison.
The experimental results are as follows:
by adopting a microbial mass spectrometry inspection technology, the escherichia coli in the article can be successfully identified and verified as shown in figure 1; sequencing results show that the PCR product contains 16S rRNA gene segments of Escherichia coli and Acinetobacter baumannii at the same time, and the gene segments are shown in a figure 2 and a figure 3; the sequencing results were entered at NCBI and the Blast alignment results are shown in FIGS. 4 and 5.
SEQUENCE LISTING
<110> Shanghai City clinical laboratory center
<120> microorganism mass spectrum detection capability verification article and preparation method thereof
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
ggttaccttg ttacgactt 19
Claims (1)
1. The application of the additive of the capability verification article in the preparation of the microorganism mass spectrum verification capability verification article is characterized in that:
the ability verification article additive is one or more purified genome DNA fragments of bacteria of different species or same species with target detection bacteria in a known current ability verification plan;
the preparation method of the microorganism mass spectrometry verifying item comprises the following steps:
(1) taking a pure culture colony of target detection bacteria or a liquid culture centrifugal precipitate of the pure culture colony in the capacity verification plan, adding absolute ethyl alcohol, and uniformly mixing to inactivate and fix the bacteria; centrifuging to remove ethanol, precipitating to room temperature, and volatilizing residual ethanol; adding sterile distilled water to prepare a bacterial liquid to obtain a basic preparation of the capacity verification article for later use;
(2) selecting bacteria of different species or different species from the same species as the target detection bacteria, performing pure culture, extracting sufficient purified genome DNA of the bacteria, measuring absorbance, adjusting concentration, packaging, and performing instant use or vacuum freeze drying; designing a pair of primers for amplifying the 16S rDNA of the bacteria to amplify the genome DNA by the design primers, adjusting the concentration of the obtained amplification product, subpackaging to obtain an additive of the capacity verification article, and immediately using or carrying out vacuum freeze drying;
(3) adding the basic preparation of the capability verification article in the step (1) into the additive of the capability verification article in the step (2), and mixing to prepare a liquid preparation of the capability verification article;
or continuously adding bovine serum albumin, freezing the liquid preparation overnight, and performing vacuum freeze drying to prepare the freeze-dried preparation of the capacity verification article.
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