Acinetobacter baumannii specific new molecular target and rapid detection method thereof

Abstract

The invention discloses an acinetobacter baumannii specific novel molecular target and a rapid detection method thereof, and provides nine novel specific molecular detection targets for identifying acinetobacter baumannii, a corresponding primer group can be designed according to target molecules, and the specific molecular target can be identified by performing PCR and SYBR fluorescent quantitative PCR technologies on a detected sample. And analyzing an electrophoresis result of the PCR product as well as an amplification curve and a Ct value of the SYBR fluorescent quantitative PCR product, so as to judge whether the acinetobacter baumannii exists or not. Compared with the existing detection means, the method can be used for detecting certain strains which have atypical biochemical reactions and are difficult to identify and distinguish through conventional biochemical identification, meanwhile, more specific molecular targets of the acinetobacter baumannii are provided, the problem of detection errors caused by insufficient discrimination of common virulence gene targets is solved, and the method is suitable for popularization and application. And the detection accuracy and reliability are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial testing, and in particular relates to a new specific molecular target of Acinetobacter baumannii and a rapid detection method thereof. Background Art

[0002] Acinetobacter baumannii is an aerobic, non-fermenting Gram-negative conditionally pathogenic coccobacillus commonly found in hospitals, often causing serious nosocomial infections. Due to its strong adaptability to the environment, Acinetobacter baumannii can form biofilms on the surfaces of various inanimate objects in hospitals, such as medical devices made of plastic, metal, glass and other materials, bed rails, door handles and other parts. The formation of biofilm not only enhances its tolerance to harsh environments, but also increases its resistance to antibiotics and disinfectants. In hospital environments, especially in intensive care units, burn departments, neurosurgery departments and other departments that admit critically ill patients for a long time, frequently use antibacterial drugs and have poor implementation of hygienic disinfection measures, the contamination trend of Acinetobacter baumannii is showing an increasingly severe trend.

[0003] With the continuous improvement of modern medical standards, invasive medical procedures are increasingly frequently used in clinical practice, and the number of detections of Acinetobacter baumannii in medical institutions has also shown a gradual upward trend. Therefore, its spread in the global medical environment is becoming increasingly severe. Due to the lack of in-depth understanding of the transmission characteristics and prevention and control points of Acinetobacter baumannii by some medical staff, they have failed to fully implement strict infection prevention and control measures in their daily work, which has increased the risk of cross-transmission of Acinetobacter baumannii between patients, between doctors and patients, and between the hospital environment and patients. The latest monitoring data released by the National Bacterial Resistance Monitoring Network on November 18, 2024 showed that during 2023, the detection rate of carbapenem-resistant Acinetobacter baumannii (CR-ABA) was as high as 55.5%, an increase of 2.1% from 53.4% in 2022, and still maintained a high level. In recent years, the number of hospital infection cases caused by Acinetobacter baumannii in some parts of my country has shown an upward trend, and has exceeded the number of cases caused by some traditional hospital infection pathogens. In the pathogenic factor system of hospital-acquired infection, the importance of Acinetobacter baumannii has become increasingly prominent, and its key position has become increasingly significant, gradually becoming one of the core elements that dominate the occurrence and development of hospital infection. In view of this, comprehensive and in-depth analysis of the hazards and risk assessment of Acinetobacter baumannii in different medical scenarios, departments, and diagnosis and treatment links has become an important task that needs to be urgently solved in the medical field.

[0004] Traditional detection methods for Acinetobacter baumannii mainly focus on microbial culture techniques and biochemical identification methods. Both have dominated past detection practices and provided a basic framework for the identification of Acinetobacter baumannii. According to the standard process of clinical microbiology testing, after sample collection, it is necessary to inoculate the sample on a specific culture medium and culture it for 24 to 48 hours or even longer in an aerobic environment at 35 to 37°C. Subsequently, a series of operations such as colony morphology observation, Gram staining, oxidase test, and biochemical reactions are carried out to determine the bacterial species. Although this method has acceptable accuracy, there are still many inherent defects. For example, its detection cycle is long, its ability to distinguish special strains is limited, it is difficult to meet the urgent need for rapid clinical diagnosis, and it is easy to cause delays in the treatment opportunity. At the same time, its operation process is relatively cumbersome and complex, and it requires high professional skills from the testing personnel, who need to have solid professional knowledge and rich practical experience. Moreover, this method is costly, involving expenses in multiple aspects such as culture media, reagents, and manpower, which increases the economic burden of detection. In addition, its sensitivity is limited, and missed detections may occur for samples with low bacterial counts or samples contaminated by other microorganisms, thus affecting the reliability of the detection results. Therefore, due to the many limitations of traditional detection methods, with the rapid development of modern medical technology, a variety of modern new molecular detection technologies have emerged continuously to make up for the deficiencies of traditional methods and meet the ever-developing needs of clinical practice and the field of microbial detection. However, the core element of the molecular diagnostic technology system lies in accurate and effective molecular detection targets. As the key hub of molecular diagnosis, it plays a decisive role in specifically identifying and detecting the nucleic acid sequences of target pathogens, directly related to the accuracy, reliability, and specificity of molecular diagnostic results, and profoundly affecting the accuracy and effectiveness of the disease diagnosis, monitoring, and research processes at the molecular level.

[0005] At present, there have been certain reports on the targets and primers for the detection of the above-mentioned Acinetobacter baumannii by molecular biology-based detection methods such as PCR technology at home and abroad. The most commonly used detection targets include blaOXA-51-like, recA, gyrB genes, etc. These target sequences have advantages such as high conservation and stable expression, and have been widely used in the identification of Acinetobacter baumannii, but there are still certain limitations. For example, as an endogenous carbapenemase gene of Acinetobacter baumannii, the blaOXA-51-like gene is extremely widely distributed in clinical isolates of Acinetobacter baumannii and can be detected in almost all such isolates. Moreover, it has high specificity and has not been found in other species. Therefore, it has currently become the most commonly used molecular marker in the field of Acinetobacter baumannii detection. However, under specific circumstances, the blaOXA-51-like gene may exist in other Acinetobacter species through horizontal gene transfer, resulting in misjudgment. In addition, this gene also exists in non-pathogenic strains and cannot distinguish highly pathogenic virulent strains from ordinary environmental strains; the recA gene is prone to cross-react with other bacterial species and has insufficient sensitivity in complex samples; the specificity and amplification efficiency of the gyrB gene need to be improved. Therefore, exploring new molecular targets with high specificity and strong sensitivity for the rapid and accurate detection of Acinetobacter baumannii has become an urgent research task in the fields of clinical microbiology and infectious diseases, which plays a crucial role in effectively curbing and precisely preventing Acinetobacter baumannii infections and improving the prognosis of patients. Summary of the Invention

[0006] In view of the above problems, the purpose of the present invention is to overcome the deficiencies of the prior art and provide a new specific molecular target for identifying Acinetobacter baumannii and its rapid detection method.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention claims a group of nucleotide sequences for identifying Acinetobacter baumannii, and the nucleotide sequences are shown as SEQ ID NO.1-9.

[0008] The said sequences are obtained by screening through bioinformatics analysis and are specific gene fragments of Acinetobacter baumannii strains.

[0009] Furthermore, the present invention also claims a primer set for identifying Acinetobacter baumannii, and the primer set is designed according to the nucleotide sequences shown as SEQ ID NO.1-9.

[0010] The product corresponding to the said primer set is all or part of the nucleotide sequences shown as SEQ ID NO.1-9.

[0011] By designing 3 sets of corresponding primer sets for PCR and 1 set of corresponding primer sets for SYBR fluorescence quantitative PCR according to the corresponding nucleotide sequences, each primer set includes a forward primer and a reverse primer, corresponding to detecting one nucleotide sequence. The amplification products of the primer sets correspond to all or part of the nucleotide sequences shown in SEQ ID NO.1-9.

[0012] As a preferred embodiment of the present invention, the nucleotide sequences of the PCR primer sets are as shown in SEQ ID NO.10-63 from 5' to 3'; wherein:

[0013] SEQ ID NO.10 and SEQ ID NO.11 are primer set 1, named the primer set of SEQ ID NO.82 sequence;

[0014] SEQ ID NO.12 and SEQ ID NO.13 are primer set 2, named the primer set of SEQ ID NO.83 sequence;

[0015] SEQ ID NO.14 and SEQ ID NO.15 are primer set 3, named the primer set of SEQ ID NO.84 sequence;

[0016] SEQ ID NO.82-84 correspond to the target of SEQ ID NO.1;

[0017] SEQ ID NO.16 and SEQ ID NO.17 are primer set 4, named the primer set of SEQ ID NO.85 sequence;

[0018] SEQ ID NO.18 and SEQ ID NO.19 are primer set 5, named the primer set of SEQ ID NO.86 sequence;

[0019] SEQ ID NO.20 and SEQ ID NO.21 are primer set 6, named the primer set of SEQ ID NO.87 sequence;

[0020] SEQ ID NO.85-87 correspond to the target of SEQ ID NO.2;

[0021] SEQ ID NO.22 and SEQ ID NO.23 are primer set 7, named the primer set of SEQ ID NO.88 sequence;

[0022] SEQ ID NO.24 and SEQ ID NO.25 are primer set 8, named the primer set of SEQ ID NO.89 sequence;

[0023] SEQ ID NO.26 and SEQ ID NO.27 are primer set 9, the primer set named for the SEQ ID NO.90 sequence;

[0024] SEQ ID NOs. 88 - 90 correspond to the target of SEQ ID NO.2;

[0025] SEQ ID NO.28 and SEQ ID NO.29 are primer set 10, the primer set named for the SEQ ID NO.91 sequence;

[0026] SEQ ID NO.30 and SEQ ID NO.31 are primer set 11, the primer set named for the SEQ ID NO.92 sequence;

[0027] SEQ ID NO.32 and SEQ ID NO.33 are primer set 12, the primer set named for the SEQ ID NO.93 sequence;

[0028] SEQ ID NOs. 91 - 93 correspond to the target of SEQ ID NO.4;

[0029] SEQ ID NO.34 and SEQ ID NO.35 are primer set 13, the primer set named for the SEQ ID NO.94 sequence;

[0030] SEQ ID NO.36 and SEQ ID NO.37 are primer set 14, the primer set named for the SEQ ID NO.95 sequence;

[0031] SEQ ID NO.38 and SEQ ID NO.39 are primer set 15, the primer set named for the SEQ ID NO.96 sequence;

[0032] SEQ ID NOs. 94 - 96 correspond to the target of SEQ ID NO.5;

[0033] SEQ ID NO.40 and SEQ ID NO.41 are primer set 16, the primer set named for the SEQ ID NO.97 sequence;

[0034] SEQ ID NO.42 and SEQ ID NO.43 are primer set 17, the primer set named for the SEQ ID NO.98 sequence;

[0035] SEQ ID NO.44 and SEQ ID NO.45 are primer set 18, the primer set named for the SEQ ID NO.99 sequence;

[0036] SEQ ID NO. 97 - 99 correspond to the target of SEQ ID NO. 6;

[0037] SEQ ID NO. 46 and SEQ ID NO. 47 are primer set 19, the primer set named with the sequence of SEQ ID NO. 100;

[0038] SEQ ID NO. 48 and SEQ ID NO. 49 are primer set 20, the primer set named with the sequence of SEQ ID NO. 101;

[0039] SEQ ID NO. 50 and SEQ ID NO. 51 are primer set 21, the primer set named with the sequence of SEQ ID NO. 102;

[0040] SEQ ID NO. 100 - 102 correspond to the target of SEQ ID NO. 7;

[0041] SEQ ID NO. 52 and SEQ ID NO. 53 are primer set 22, the primer set named with the sequence of SEQ ID NO. 103;

[0042] SEQ ID NO. 54 and SEQ ID NO. 55 are primer set 23, the primer set named with the sequence of SEQ ID NO. 104;

[0043] SEQ ID NO. 56 and SEQ ID NO. 57 are primer set 24, the primer set named with the sequence of SEQ ID NO. 105;

[0044] SEQ ID NO. 103 - 105 correspond to the target of SEQ ID NO. 8;

[0045] SEQ ID NO. 58 and SEQ ID NO. 59 are primer set 25, the primer set named with the sequence of SEQ ID NO. 106;

[0046] SEQ ID NO. 60 and SEQ ID NO. 61 are primer set 26, the primer set named with the sequence of SEQ ID NO. 107;

[0047] SEQ ID NO. 62 and SEQ ID NO. 63 are primer set 27, the primer set named with the sequence of SEQ ID NO. 108;

[0048] SEQ ID NO. 106 - 108 correspond to the target of SEQ ID NO. 9.

[0049] The nucleotide sequences of the SYBR fluorescence quantitative PCR primer sets are shown as SEQ ID NO. 64 to 81 from 5' to 3'; among them:

[0050] SEQ ID NO. 64 and SEQ ID NO. 65 are primer set 28, the primer set named SEQ ID NO. 109 sequence, corresponding to

[0051] the target of SEQ ID NO. 1;

[0052] SEQ ID NO. 66 and SEQ ID NO. 67 are primer set 29, the primer set named SEQ ID NO. 110 sequence, corresponding to

[0053] the target of SEQ ID NO. 2;

[0054] SEQ ID NO. 68 and SEQ ID NO. 69 are primer set 30, the primer set named SEQ ID NO. 111 sequence, corresponding to

[0055] the target of SEQ ID NO. 3;

[0056] SEQ ID NO. 70 and SEQ ID NO. 71 are primer set 31, the primer set named SEQ ID NO. 112 sequence, corresponding to

[0057] the target of SEQ ID NO. 4;

[0058] SEQ ID NO. 72 and SEQ ID NO. 73 are primer set 32, the primer set named SEQ ID NO. 113 sequence, corresponding to

[0059] the target of SEQ ID NO. 5;

[0060] SEQ ID NO. 74 and SEQ ID NO. 75 are primer set 33, the primer set named SEQ ID NO. 114 sequence, corresponding to

[0061] the target of SEQ ID NO. 6;

[0062] SEQ ID NO. 76 and SEQ ID NO. 77 are primer set 34, the primer set named SEQ ID NO. 115 sequence, corresponding to

[0063] the target of SEQ ID NO. 7;

[0064] SEQ ID NO.78 and SEQ ID NO.79 are primer set 35, the primer set named after the sequence of SEQ ID NO.116, corresponding to

[0065] the target of SEQ ID NO.8;

[0066] SEQ ID NO.80 and SEQ ID NO.81 are primer set 36, the primer set named after the sequence of SEQ ID NO.117, corresponding to the target of SEQ ID NO.9.

[0067] Furthermore, the present invention also claims the application of the primer set in identifying Acinetobacter baumannii.

[0068] The present invention also provides a method for identifying Acinetobacter baumannii using the PCR primer set, comprising the following steps:

[0069] A1: Perform PCR amplification on the DNA of the sample to be tested using one of the primer sets as described;

[0070] A2: Perform agarose gel electrophoresis to detect the amplification product;

[0071] A3: Observe whether the amplification product meets the expectation. If a corresponding single amplification band appears in the electrophoresis result, it is determined that the target bacteria are contained in the sample, otherwise it is determined that the target bacteria are not contained in the sample.

[0072] Generally, only one set of primers is contained in a PCR system; by setting up multiple PCR systems, the DNA of a single bacterium can be amplified using different primers simultaneously to improve the detection efficiency. The product corresponding to the primer of the present invention has good specificity, and it can be determined whether Acinetobacter baumannii exists by observing whether the amplification product is at the expected position.

[0073] As a preferred embodiment of the present invention, the PCR amplification system in A1 includes 2×PCR Mix, template DNA, primer set, and ddH2O.

[0074] As a preferred embodiment of the present invention, the PCR amplification system is: 2×PCR Mix 10 μL, template DNA 0.8 μL, 0.8 μL each of upstream and downstream primers, and ddH2O is added to make up the volume to 20 μL.

[0075] As a preferred embodiment of the present invention, the PCR amplification program in A1 is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 30 s; denaturation, annealing, and extension are carried out for 35 cycles in total; finally, extension at 72°C for 5 min.

[0076] Furthermore, the present invention also provides a method for identifying Acinetobacter baumannii using the SYBR fluorescence quantitative PCR primer set, comprising the following steps:

[0077] B1: Obtain the DNA of the sample to be tested. The genomic DNA is extracted from a bacterial DNA extraction kit (Tiangen Biochemical, China);

[0078] B2: Measure the concentration of the DNA in S1, convert it into the copy number through a formula; and perform 10-fold serial dilution on the DNA in S1 to obtain a DNA dilution solution. Select the 10 0 ~10 -7 -fold dilution solution of DNA as the standard sample for preparing the standard curve;

[0079] B3: Use one of the primer sets described in claim 2 or 4 to perform SYBR fluorescence quantitative PCR amplification on the DNA of the sample to be tested, and detect the amplification curve and CT value of Acinetobacter baumannii DNA in the standard sample. If the amplification curve is S-shaped and the Ct value ≤ 35, it is determined as positive; if the Ct value ≥ 40, it is determined as negative; if the Ct value is between 37 - 40, perform a repeated experiment. If the repeated result shows that the Ct value < 40 and the amplification curve has an obvious peak, it is determined as positive; otherwise, it is negative.

[0080] As a preferred embodiment of the present invention, the SYBR fluorescence quantitative PCR amplification system in B3 includes 2×Q3SYBR PCR Master Mix, template DNA, primer set, and ddH2O.

[0081] As a preferred embodiment of the present invention, the PCR amplification system is: 2×Q3 SYBR PCR Master Mix 10 μL, template DNA 1 μL, each of the upstream and downstream primers 0.4 μL, and ddH2O is added to make up the volume to 20 μL.

[0082] As a preferred embodiment of the present invention, the SYBR fluorescence quantitative PCR amplification program in B3 is: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing and extension at 60°C for 30 s; denaturation, annealing, and extension are carried out for 40 cycles in total.

[0083] The beneficial effects of the present invention are:

[0084] The present invention discloses nine specific molecular targets for identifying Acinetobacter baumannii, related primer sets, and corresponding PCR and fluorescence quantitative PCR detection methods. Compared with existing detection means, the present invention can detect the presence of Acinetobacter baumannii in the target detection substance without preparing monoclonal antibodies against Acinetobacter baumannii, reducing the detection cost; at the same time, the detection method of the present invention provides more specific molecular targets for Acinetobacter baumannii, improving the detection error problem caused by insufficient discrimination of common virulence gene targets, enhancing the accuracy and reliability of detection, and having higher practical value; the method of the present invention also has the advantages of simple and rapid operation, strong specificity of detection results, high sensitivity, low detection cost, and intuitive and clear result determination, which is of great significance in the fields of medical inspection and pathogen prevention and control, especially for the rapid and accurate identification of Acinetobacter baumannii in clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 It is the electrophoresis result for the feasibility evaluation of the PCR detection method for Acinetobacter baumannii in Example 2;

[0086] Figure 2 It is the electrophoresis result (partial) for the sensitivity evaluation of the PCR detection method for Acinetobacter baumannii in Example 3;

[0087] Figure 3 It is the electrophoresis result (partial) for the sensitivity evaluation of the PCR detection method for Acinetobacter baumannii in Example 3;

[0088] Figure 4 It is the electrophoresis result (partial) for the sensitivity evaluation of the PCR detection method for Acinetobacter baumannii in Example 3;

[0089] Figure 5 It is the amplification curve, melting curve, and standard curve (partial) for the sensitivity evaluation of the SYBR fluorescence quantitative PCR detection method for Acinetobacter baumannii in Example 3.

[0090] Figure 6 It is the amplification curve, melting curve, and standard curve (partial) for the sensitivity evaluation of the SYBR fluorescence quantitative PCR detection method for Acinetobacter baumannii in Example 3.

[0091] Figure 7 It is the electrophoresis result for the specificity evaluation of the PCR detection method for Acinetobacter baumannii in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0092] In order to more concisely and clearly show the technical solutions, objectives, and advantages of the present invention, the following further detailed description of the present invention is made in conjunction with specific embodiments and their accompanying drawings.

[0093] Example 1: Mining of Specific New Molecular Targets for Acinetobacter baumannii

[0094] I. Isolation and preservation of Acinetobacter baumannii

[0095] Acinetobacter baumannii strains were isolated from clinical samples such as sputum, alveolar lavage fluid, drainage fluid, wound secretions, bile, catheters, and throat swabs from patients of all age groups and different genders in the hospital, with a total of 174 samples. Under aseptic conditions, 0.1 g of solid strain was inoculated into Columbia blood agar medium (Chromagar, China), cultured at 37 °C for 18 - 24 h, and then single colonies in zone 3 were scraped with a filter paper strip under aseptic conditions and stored in a 1.5 mL centrifuge tube, and stored in the laboratory refrigerator. After mass spectrometry identification, among the 174 Acinetobacter baumannii strains isolated from clinical samples, 152 were indeed Acinetobacter baumannii and 20 were non - Acinetobacter baumannii. The 152 Acinetobacter baumannii strains were cultured in broth medium, transferred, and preserved in a - 20 °C low - temperature refrigerator with 30% glycerol.

[0096] II. Identification of Acinetobacter baumannii

[0097] Bacterial DNA was extracted using a bacterial DNA extraction kit (Tiangen Biochemical, China), and then PCR amplification was carried out using 2×PCR mix (Dongsheng Biotech). The PCR amplification primers used were universal primers for the 16S rRNA gene. The PCR reaction conditions were: pre - denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s; annealing at 60 °C for 30 s; extension at 72 °C for 30 s; denaturation, annealing, and extension were carried out for 35 cycles in total; and finally, extension at 72 °C for 5 min. After gel extraction and recovery of the PCR products, first - generation sequencing was carried out. After identification, among the 174 Acinetobacter baumannii strains isolated from clinical samples, 152 were indeed Acinetobacter baumannii. The nucleotide sequence of Acinetobacter baumannii required to be protected by this patent was compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov), and the results showed that it had the highest homology with Acinetobacter baumannii. For strains with both Identity and Coverage in the comparison results being more than 99% similar to the known Acinetobacter baumannii, they could be determined as Acinetobacter baumannii.

[0098] III. Discovery and verification of specific new molecular targets of Acinetobacter baumannii

[0099] Based on the GenBank database and the whole - genome DNA sequence of Acinetobacter baumannii self - tested by this team, bioinformatics analysis was carried out; 55 non - essential genes unique to Acinetobacter baumannii strains were screened, and further analysis and screening yielded 9 specific gene fragments of Acinetobacter baumannii. Three pairs of specific primer sets were designed for each gene fragment, and the nucleotide sequences of the gene fragments are shown as SEQ ID NO.1 - 9.

[0100] Example 2: Establishment of a rapid detection method for new molecular targets of Acinetobacter baumannii

[0101] (1) Primer design

[0102] Based on the Acinetobacter baumannii specific detection targets SEQ ID NO.1 - 9 described in Example 1, use Oligo software to design a specific PCR amplification primer set SEQ ID NO.10 - 63 (including forward primers and reverse primers), and the primer set sequences are shown in Table 1.

[0103] Table 1: Sequences of the primer set for Acinetobacter baumannii specific PCR detection

[0104]

[0105]

[0106]

[0107] Based on the Acinetobacter baumannii specific detection targets SEQ ID NO.1 - 9 described in Example 1, use Oligo software to design a specific SYBR fluorescence quantitative PCR amplification primer set SEQ ID NO.64 - 81 (including forward primers and reverse primers), and the primer set sequences are shown in Table 2.

[0108] Table 2: Sequences of the primer set for Acinetobacter baumannii specific SYBR fluorescence quantitative PCR detection

[0109]

[0110]

[0111] (2) Method for identifying Acinetobacter baumannii, the steps are as follows:

[0112] A. DNA template preparation: Incubate the test strains in LB liquid medium for enrichment culture, and use a bacterial DNA extraction kit (Tiangen Biochemical, China) to extract their bacterial genomic DNA respectively as the test templates.

[0113] B. PCR detection system and amplification program:

[0114] Extract the genomic genes of the test microorganisms using a bacterial DNA extraction kit (Tiangen Biochemical, China), and then add them to the PCR detection reaction system for Acinetobacter baumannii. The PCR detection system for Acinetobacter baumannii is as follows:

[0115]

[0116] The PCR amplification program is as follows:

[0117]

[0118] C. Perform agarose gel electrophoresis on the PCR amplification products.

[0119] D. Observe whether there is a single amplification band at the position corresponding to the size of the products of each primer set. If the electrophoresis result shows a single amplification band at the target band for the amplification products, it indicates that the sample contains Acinetobacter baumannii corresponding to the detection; if no corresponding single amplification band appears, the sample does not contain Acinetobacter baumannii.

[0120] E. SYBR fluorescence quantitative PCR detection system and amplification program:

[0121] Extract the genomic genes of the microorganism to be tested using a bacterial DNA extraction kit (Tiangen Biochemical, China), and then add them to the SYBR fluorescence quantitative PCR detection reaction system for Acinetobacter baumannii. The SYBR fluorescence quantitative PCR detection system for Acinetobacter baumannii is as follows:

[0122] 2×Q3 SYBR PCR Master Mix

[0123] Forward primer (10 μmol / L)

[0124] Reverse primer (10 μmol / L)

[0125] Template DNA A (20.4 ng / μL)

[0126] ddH2O

[0127] In the SYBR fluorescence quantitative PCR detection reaction system, the SYBR fluorescence quantitative PCR detection system for Acinetobacter baumannii is as follows:

[0128] 10 μL

[0129] 0.4 μL

[0130] 0.4 μL

[0131] 1.0 μL

[0132] 8.2 μL

[0133] The SYBR fluorescence quantitative PCR amplification program is as follows:

[0134]

[0135] Melting curve analysis:

[0136] 65°C 5 s

[0137] 95°C 0.5°C / s

[0138] F. Read the SYBR fluorescence quantitative PCR amplification curve and Ct value of Acinetobacter baumannii DNA in the standard sample. If the amplification curve is S-shaped and the Ct value ≤ 35, it is judged as positive; if the Ct value ≥ 40, it is judged as negative; if the Ct value is between 37 - 40, repeat the experiment. If the Ct value of the repeated result is < 40 and the amplification curve has an obvious peak, it is judged as positive; otherwise, it is negative.

[0139] (3) Verification of the feasibility of primer sets

[0140] Using the method described in step (2) above, take 1 clinical isolate of Acinetobacter baumannii to detect the primer sets SEQ ID NO.10 - 63 described in step (1). Among them, A. DNA template preparation is to extract the genomic DNA of Acinetobacter baumannii. Set a blank control, and the template of the blank control is an aqueous solution without genomic DNA. The electrophoresis results are as shown in the appendix Figure 1 as shown, and the result interpretation is shown in Table 3.

[0141] Table 3: Detection results of the feasibility verification of Acinetobacter baumannii primer sets

[0142]

[0143]

[0144]

[0145] From Figure 1 and Table 3, it can be seen that the electrophoresis results of 27 primer sets all show specific amplification bands, proving that the primers designed in step (1) above are all primers for specific new molecular targets of Acinetobacter baumannii.

[0146] Example 3: Evaluation of the sensitivity and specificity of a rapid detection method for specific new detection targets of Acinetobacter baumannii

[0147] (1) Results of the sensitivity evaluation of the new molecular target PCR detection method for Acinetobacter baumannii

[0148] Using the method described in Example 2 (primer sets SEQ ID NO.10 - 63), dilute the genomic DNA of Acinetobacter baumannii with a concentration of 20.4 ng / μL in pure water according to a 10-fold gradient to obtain concentrations of 20.4×10 -1 、20.4×10 -2 、20.4×10 -3 、20.4×10 -4 、20.4×10 -5 、20.4×10 -6 、20.4×10 -7Genomic DNA serially diluted at a gradient of ng / μL. A blank control was set, and the template for the blank control was an aqueous solution without genomic DNA. The electrophoresis results are shown in the appendix Figures 2 - 4 as follows, where M is the marker, and M15(1), M15(2), and M15(3) are the same marker.

[0149] (2) Evaluation results of the sensitivity of the SYBR fluorescence quantitative PCR detection method for the new molecular target of Acinetobacter baumannii

[0150] Using the method described in Example 2 (primer set SEQ ID NO.64 - 81), the genomic DNA of Acinetobacter baumannii with a concentration of 20.4 ng / μL was serially diluted at a 10-fold gradient with pure water to obtain genomic DNA serially diluted at a gradient of 20.4×10 -1 、20.4×10 -2 、20.4×10 -3 、20.4×10 -4 、20.4×10 -5 、20.4×10 -6 、20.4×10 -7 ng / μL. A blank control was set, and the template for the blank control was an aqueous solution without genomic DNA. The amplification curves and melting curves of the SYBR fluorescence quantitative PCR products are shown in the appendix Figures 5 - 6 as follows, and a standard curve was prepared based on the Ct values and DNA concentrations.

[0151] (3) Evaluation results of the specificity of the PCR detection method for the new molecular target of Acinetobacter baumannii

[0152] One strain of Acinetobacter baumannii and 22 strains of non-Acinetobacter baumannii (one strain each of Acinetobacter calcoaceticus, Acinetobacter lwoffii, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter johnsonii, Acinetobacter radioresistens, Staphylococcus warneri, Streptococcus agalactiae, Staphylococcus capitis, Staphylococcus saprophyticus, Enterococcus gallinarum, Streptococcus constellatus subsp. constellatus, Enterococcus avium, Streptococcus gallolyticus, Staphylococcus cohnii subsp. urealyticum, Cupriavidus sp., Listeria monocytogenes, Bacillus cereus, Salmonella enterica subsp. enterica serovar Typhimurium, Cronobacter sakazakii, Staphylococcus aureus, Escherichia coli) were subjected to PCR detection according to the method of Example 2. Among them, A. DNA template preparation was to extract the genomic DNA of each bacterium respectively. A blank control was set, and the template for the blank control was an aqueous solution without genomic DNA.

[0153] The strains of each bacterium used and the detection results are shown in Table 4 below. In the table, in the detection result column, "+" indicates positive and "-" indicates negative. The electrophoresis results of the PCR products are as Figure 7 shown.

[0154] Table 4: Results of the specificity evaluation test for the detection of Acinetobacter baumannii in the present invention

[0155] Serial number Strain name Type Strain number Number of strains Result 1 Acinetobacter baumannii Acinetobacter baumannii Clinical isolate 1 + 2 Acinetobacter calcoaceticus Acinetobacter calcoaceticus Clinical isolate 1 - 3 Acinetobacter lwoffi Acinetobacter lwoffi Clinical isolate 1 - 4 Acinetobacter haemolytius Acinetobacter haemolytius Clinical isolate 1 - 5 Acinetobacter junii Acinetobacter junii Clinical isolate 1 - 6 Acinetobacter johnsonii Acinetobacter johnsonii Clinical isolate 1 - 7 Acinetobacter radioresistens Acinetobacter radioresistens Clinical isolate 1 - 8 Staphylococcus warneri Staphylococcus warneri Clinical isolate 1 - 9 Streptococcus agalactiae Streptococcus agalactiae Clinical isolate 1 - 10 Staphylococcus capitis Staphylococcus capitis Clinical isolate 1 - 11 Staphylococcus saprophyticus Staphylococcus saprophyticus Clinical isolate 1 - 12 Enterococcus gallinarum Enterococcus gallinarum Clinical isolate 1 - 13 S.constellatus subsp.constellatus Streptococcus constellatus subsp. constellatus Clinical isolate 1 - 14 Enterococcus faecium Enterococcus faecium Clinical isolate 1 - 15 Streptococcus gallolyticus Streptococcus gallolyticus Clinical isolate 1 - 16 Staphylococcus saprophyticus Staphylococcus saprophyticus subsp. ureolyticus Clinical isolate 1 - 17 Cupriavidus Cupriavidus Clinical isolate 1 - 18 Listeria monocytogenes Listeria monocytogenes ATCC 19115 1 - 19 Bacillus cereus Bacillus cereus ATCC 11778 1 - 20 Salmonella enterica subsp.enterica Salmonella enterica subsp. enterica ATCC 13076 1 - 21 Cronobacter sakazakii Cronobacter sakazakii ATCC 29544 1 - 22 Staphylococcus aureus subsp Staphylococcus aureus ATCC 29213 1 - 23 Escherichia coli Escherichia coli ATCC 700728 1 -

[0156] From Figure 3 and Table 4, it can be seen that only Acinetobacter baumannii shows specific amplification bands, and no specific bands are shown in other non-Acinetobacter baumannii, indicating that the method of the present invention has high specificity.

Claims

1. An isolated nucleotide, characterized in that, It has a sequence as shown in any one of SEQ ID NO.1-9.

2. Use of the nucleotide according to claim 1 in the preparation of a reagent for differentiating Acinetobacter baumannii for non-therapeutic and diagnostic purposes.

3. A primer pair, characterized in that, It is used to specifically amplify the nucleotide according to claim 1.

4. The primer pair according to claim 3, characterized in that, It has sequences such as SEQ ID NO.10+x and SEQ ID NO.11+x; where x is any even number taken from 0 to 52.

5. The primer pair according to claim 3, characterized in that, It has sequences such as SEQ ID NO.10+x and SEQ ID NO.11+x; where x is any even number taken from 54 to 70.

6. A method for differentiating Acinetobacter baumannii for non-therapeutic and non-diagnostic purposes, characterized in that, It includes the following steps: Using one of the primer pairs according to claim 3, performing PCR amplification on the DNA of the sample to be tested; if the corresponding amplification product appears in the electrophoresis result, it is determined that the target bacteria are contained in the sample, otherwise it is determined that the target bacteria are not contained in the sample.

7. The method according to claim 6, wherein The PCR amplification system includes 10 μL of 2×PCR Mix, 0.8 μL of template DNA, 0.8 μL of each of the upstream and downstream primers, and ddH2O is used to make up the volume to 20 μL; the PCR amplification program is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 30 s; denaturation, annealing, and extension are carried out for 35 cycles in total; finally, extension at 72°C for 5 min.

8. A method for differentiating Acinetobacter baumannii for non-therapeutic and non-diagnostic purposes, characterized in that, It includes the following steps: Using one of the primer pairs according to claim 4, performing fluorescence quantitative PCR amplification on the DNA of the sample to be tested, detecting the amplification curve and CT value of Acinetobacter baumannii DNA in the standard sample. If the amplification curve is S-shaped and the Ct value ≤ 35, it is determined to be positive; if the Ct value ≥ 40, it is determined to be negative; if the Ct value is between 37 and 40, a repeated experiment is carried out. If the repeated result shows that the Ct value < 40 and the amplification curve has an obvious peak, it is determined to be positive; otherwise, it is negative.

9. The method according to claim 8, characterized in that, The PCR amplification system is: 10 μL of 2×Q3 SYBR PCR MasterMix, 1 μL of template DNA, 0.4 μL of each of the upstream and downstream primers, and ddH2O is used to make up the volume to 20 μL.

10. The method according to claim 8, characterized in that, The fluorescence quantitative PCR amplification program is: pre-denaturation at 95°C for 30 s; Denaturation at 95°C for 10 s; annealing and extension at 60°C for 30 s; denaturation, annealing, and extension are carried out for 40 cycles in total.

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