阅读说明：本技术 一种鼠疫耶尔森氏菌蛋白质谱库的建立方法 (Method for establishing Yersinia pestis protein library ) 是由 石丽媛 冯彬 王鹏 张海鹏 杨丰义 钟佑宏 董珊珊 谭红丽 郭英 段存娟 杨丽华 于 2020-04-10 设计创作，主要内容包括：本发明公开一种鼠疫耶尔森氏菌蛋白质谱库的建立方法,属于微生物鉴定技术领域。本发明所述方法将鼠疫耶尔森氏菌质谱图加入到蛋白质谱仪中,并通过引入二级算法,成功的将鼠疫耶尔森氏菌和假结核耶尔森氏菌鉴别区分开,使用该方法得到的质谱仪能够准确鉴定鼠疫耶尔森氏菌。(The invention discloses a method for establishing a Yersinia pestis protein library, belonging to the technical field of microorganism identification. The method adds the Yersinia pestis mass spectrogram into a protein spectrometer, successfully distinguishes the Yersinia pestis and the Yersinia pseudotuberculosis by introducing a secondary algorithm, and the mass spectrometer obtained by the method can accurately identify the Yersinia pestis.)

1. A method for establishing a Yersinia pestis protein library is characterized by comprising the following steps:

(1) selecting a library-establishing strain: selecting more than 3 strains of different epidemic areas, wherein the strain of each epidemic area is more than or equal to 5 strains, and correcting the quality control by using Escherichia coli E.coli ATCCC 8739;

(2) resuscitating, subculturing and largely culturing the selected strain, simultaneously performing plague phage lysis test to ensure that the strain is not polluted, then extracting protein liquid, and performing a sterile test on the bacterial protein liquid;

(3) point plate and map collection: after the aseptic experiment proves that no Yersinia pestis survives, spotting the bacterial protein liquid on a plate; each strain of the bacterial protein liquid is spotted with 15 target spots in parallel, the bacterial protein liquid is spotted at the positions of the 15 target spots outside the quality control bacteria, and each target spot acquires the atlas for 2 times; thus, each strain generates 30 maps;

(4) building a library: building a library of all Yersinia pestis strains selected in the step (1), wherein each strain has 30 maps, overlapping the maps, removing the maps with special peak shapes, and storing all the remaining maps in the library, wherein the system automatically generates a protein library of the strain;

(5) introducing a two-stage algorithm

First-stage retrieval: preprocessing the spectrogram, searching a database containing Yersinia pestis and Yersinia pseudotuberculosis by using a search identification algorithm based on spectrogram similarity matching, sequencing from high to low according to spectrogram comparison similarity scores, and taking out the first 10 results;

judging secondary retrieval conditions: in the first 10 results, if the 1 st result is any bacterium or more than 6 results are any bacteria, the second-level search is started, and if the above conditions are not met, the first result is directly output as the final identification result;

(6) two-level search execution

Carrying out normalization transformation based on the strongest peak on the acquired spectrogram and a database standard spectrogram, wherein the highest peak is 1 after transformation;

secondly, carrying out Cartesian coordinate projection on all protein peaks of an unknown spectrogram to be identified, wherein the abscissa is a mass-to-charge ratio, and the ordinate is relative intensity after normalization;

searching the projection in the step two by taking 3063Da as a center and a relative error of +/-500 ppm, and accumulating all the projection signals searched in the range to obtain a projection accumulated value S;

judging S, if S is more than or equal to 0.2, judging that the identification result is Yersinia pestis, otherwise, judging that the identification result is Yersinia pseudotuberculosis, and finishing the retrieval.

2. The method for establishing a Yersinia pestis protein library according to claim 1, wherein: the culture conditions in the step (2) are as follows: LB plus blood medium, 28 ℃ for 24 hours.

3. The method for establishing a Yersinia pestis protein library according to claim 1, wherein: and (3) preparing the bacterial protein liquid by adopting an ethanol/formic acid extraction method in the step (2).

Technical Field

The invention relates to a method for establishing a Yersinia pestis protein library, belonging to the technical field of microorganism identification.

Background

Plague is a natural epidemic disease caused by Yersinia pestis, and seriously harms human health. The infectious disease control law of China lists plague as a class A infectious disease. Historically, three worldwide pandemics of plague have occurred, causing the death of hundreds of millions of people; in 8 months in 2017, outbreak of plague outbreaks of african motor gaska, 2600 cases of cumulative report cases and 200 cases of death; in 2019, Beijing in 11 months confirmed diagnosis of 2 cases of plague of lung from Nemontmoringa tinctoria, and in the month, Beijing and Guizhou check-up confirmed diagnosis of 2 cases of plague of gland in turn. The threat of plague to humans still exists.

With the rapid development of science and technology, protein mass spectrometry technology has been widely applied to the identification of microorganisms, and the yersinia pestis, which has the greatest threat to human beings, in yersinia has not been banked in a mass spectrometer and cannot be accurately identified. For example, the M-Discover 100 mass spectrometer developed by Meihua medical science and technology Limited in the Jeans, which has a Cure of 5159 strains in the bacterial field and 37 strains in the Yersinia genus, including Yersinia such as pseudotuberculosis, enterocolitis, Roche and Lu's, and Yersinia pestis has not been pooled in the mass spectrometer.

The reason why the yersinia pestis is not put in storage is that the yersinia pestis is highly pathogenic, and the activity of the yersinia pestis needs to be carried out in a high-grade biosafety laboratory, so that the yersinia pestis is limited to be obtained and used by a general organization or a company, and the capability of a mass spectrometer for identifying the yersinia pestis is also limited. On the other hand, the inventor experimentally found that, when the test strain is yersinia pestis before the yersinia pestis is banked, the identification result is yersinia pseudotuberculosis (yersinia pseudotuberculosis is the closest species to yersinia pestis); after the Yersinia pestis is built, the Yersinia pestis can be successfully identified, but the Yersinia pseudotuberculosis is incorrectly identified and is identified as Yersinia pestis. Yersinia currently exists in 18 species, of which there are 3 species that are pathogenic to humans: yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. Yersinia pestis is considered to be evolved from Yersinia pseudotuberculosis, the similarity of genomes of the Yersinia pestis and the Yersinia pseudotuberculosis even exceeds the similarity of strains in certain bacteria, but the difference is large in pathogenicity of the bacteria, the Yersinia pestis can cause bubonic plague, pulmonary plague and the like, patients can be attacked acutely, the Yersinia pestis can die quickly when no effective antibiotic is used for treating, the pulmonary plague can be spread through droplets, and the large-area epidemic of the disease can be easily caused under the condition of no respiratory protection. While infection with Yersinia pseudotuberculosis, patients mainly present with intestinal infections and erythema and nodules of the skin. Therefore, the two Yersinia species are not harmful to human beings, and have serious consequences once the identification fails.

In conclusion, the incorporation of the yersinia pestis protein mass spectrum into the mass spectrometer library not only requires the existence of strain sources and the preparation of corresponding biosafety laboratories, but also has the technical problem, and the key to the technical problem is how to solve the problem of identification confusion between yersinia pestis and yersinia pseudotuberculosis, and how to further distinguish the yersinia pestis from the yersinia pseudotuberculosis.

Disclosure of Invention

The invention aims to provide a method for establishing a Yersinia pestis protein library, which specifically comprises the following steps:

(1) selecting a library-establishing strain: selecting more than 3 strains of different epidemic areas, wherein the strain of each epidemic area is more than or equal to 5 strains, and correcting the quality control by using Escherichia coli E.

(2) Reviving, subculturing and largely culturing the selected strain, simultaneously performing plague phage lysis test to ensure that the strain is not polluted, extracting protein liquid, and performing aseptic test on the bacterial protein liquid.

(3) Point plate and map collection: after the Yersinia pestis survives in the aseptic experiment, spotting the bacterial protein liquid on a plate; each strain of the bacterial protein liquid is spotted with 15 target spots in parallel, the bacterial protein liquid is spotted at the positions of the 15 target spots outside the quality control bacteria, and each target spot acquires the atlas for 2 times; thus, each strain generates 30 maps in total.

(4) Building a library: building a library of 16 Yersinia pestis strains, wherein each strain has 30 maps, overlapping the maps, removing the maps with special peak shapes, and storing the rest maps in the library, wherein the system automatically generates a protein library of the strains.

(5) Introducing a two-stage algorithm

First-stage retrieval: after the spectrogram is preprocessed, a retrieval identification algorithm based on spectrogram similarity matching is adopted to retrieve the databases containing the Yersinia pestis and the Yersinia pseudotuberculosis, similarity scores are compared according to the spectrogram and are ranked from high to low, and the top 10 results are taken out.

Judging secondary retrieval conditions: in the first 10 results, if the 1 st result is any bacterium or more than 6 results are any bacteria, the second-level search is started, and if the above conditions are not met, the first result is directly output as the final identification result.

(6) Two-level search execution

Carrying out normalization transformation based on the strongest peak on the acquired spectrogram and the database standard spectrogram, wherein the highest peak is 1 after transformation.

Secondly, carrying out Cartesian coordinate projection on all protein peaks of the unknown spectrogram to be identified, wherein the abscissa is the mass-to-charge ratio, and the ordinate is the relative intensity after normalization.

And thirdly, searching the projection in the step two by taking 3063Da as a center and a relative error of +/-500 ppm, and accumulating all the projection signals searched in the range to obtain a projection accumulated value S.

Judging S, if S is more than or equal to 0.2, judging that the identification result is Yersinia pestis, otherwise, judging that the identification result is Yersinia pseudotuberculosis, and finishing the retrieval.

The culture conditions in the step (2) of the invention are as follows: LB plus blood medium, 28 ℃ for 24 hours.

In step (2), the bacterial protein solution is prepared by an ethanol/formic acid extraction method.

The invention has the beneficial effects that: the invention discovers for the first time that in protein mass spectrum detection, the yersinia pestis and the yersinia pseudotuberculosis are easy to be confused, and the introduction of a secondary algorithm successfully distinguishes the yersinia pestis and the yersinia pestis, so that the protein mass spectrometer can identify the yersinia pestis, and the established yersinia pestis protein spectrum library has high detection accuracy.

Drawings

FIG. 116 shows the location and information in the protein spectrometer library list after the library was constructed from Yersinia pestis;

FIG. 2 illustrates the coating position of quality control bacterium Escherichia coli;

FIG. 3 protein profile of Yersinia pestis (EV76, 72, 1408, 1409);

FIG. 4 protein spectra of Yersinia pseudotuberculosis (PST1, PST 2).

Detailed Description

The present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited to the above description.

There are 12 natural plague areas in China, and Yunnan has 3 types of natural plague areas, namely a natural plague area of the Musca domestica, a natural plague area of the Caragana glauca and a natural plague area of the Musca domestica in Lijiang. In the embodiment of the invention, the project relying unit reserves Yersinia pestis species resources from the last 50 th generation to the present, and strains in various epidemic areas are preserved.