A primer, probe combination and detection kit for identifying Ochrobacterium hominis

Abstract

The present invention relates to a primer, probe combination and detection kit for identifying Ochrobacterium hominis. The present invention adopts a one-step fluorescence quantitative PCR detection technology with three genes and dual fluorescent group labels, which can detect and identify nucleic acid DNA of Ochrobacterium genus and Ochrobacterium hominis species and strains in pure bacteria and blood and other specimens, with high sensitivity, and can detect 3.67fg of nucleic acid DNA (Ct value is 35.38), that is, the PCR reaction system can detect as long as there is 1 nucleic acid DNA copy number. The three genes are used for simultaneous detection, which greatly improves the detection rate of nucleic acid DNA of Ochrobacterium genus and Ochrobacter hominis in blood and other specimens. The kit is simple to operate, does not require gel electrophoresis, and after the bacterial genomic DNA of the specimen is extracted, the detection and identification results can be reported in 1 hour, which is particularly suitable for the detection and identification of trace nucleic acid DNA in blood and other specimens.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and relates to a specific nucleotide site for identifying Ochrobacterium hominis, a primer and probe combination for the site, and an application of the primer and probe combination in identifying Ochrobacterium hominis products, and specifically relates to a kit for identifying Ochrobacterium hominis. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Ochrobacterium and Ochrobacter hominis strains are non-fermenting Gram-negative aerobic rods with a wide distribution and are present in soil, water and other environments. When the local or systemic immune function of the human body is reduced, it can cause infection, and can cause sepsis, meningitis, peritonitis and other diseases. In recent years, literature has reported that Ochrobacter hominis can cause illness in people with normal immune function, so the bacteria is also a human pathogen. Some strains are very virulent and can cause local abscesses or even septic shock. It is this neglected pathogen that has frequently appeared in clinical cases in recent years. The increase in detection rate is due to the widespread use of fully automatic microbial analysis systems in bacterial culture identification and drug sensitivity testing in clinical microbiology laboratories. These instruments include: BACT / ALERT 3D fully automatic blood culture instrument (BioMérieux, France), Vitek 2compact fully automatic microbial identification system, automatic bacterial identification system Vitek 2compact bacterial analyzer and matching bacterial identification plate GN card (BioMérieux, France), and BioMérieux API20 NE identification strips. However, Ochrobacter hominis is easily misdiagnosed as Brucella, Bordetella bronchiseptica and Moraxella phenylpyruvicola, as well as misjudgment between species within the genus Ochrobacter. Among them, Ochrobacter hominis has the highest error rate. The genetic characteristics, biochemical characteristics, culture characteristics and serological characteristics of Ochrobacter hominis have many similarities with Brucella, which increases the difficulty of clinical detection and identification.

[0004] With the development of molecular biology technology, PCR method has been used in auxiliary diagnosis of diseases and detection of pathogens. 16S rRNA gene sequencing method based on PCR amplification is widely used in molecular biological bacterial classification of microorganisms. Fluorescence quantitative PCR technology is a nucleic acid qualitative and quantitative detection technology that integrates PCR technology, fluorescent signal detection and data analysis. It has high specificity, sensitivity, and the advantages of being fast and simple. For the 8 classic strains of Ochrobactrum, the 16S rRNA gene has a high degree of consistency. The 8 strains of Ochrobactrum include: Ochrobactrum anthropi (human Ochrobactrum), Ochrobactrum pituitosa, Ochrobactrum intermedia, Ochrobactrum pseudogrignonense, Ochrobactrum soli, Ochrobactrum thiophenivorans, Ochrobactrum tritici, Ochrobactrum] quorum nocens. Among them, Ochrobactrum anthropi, which is pathogenic to humans, has a very high similarity with Ochrobactrum intermedia strains, which causes technical difficulties in clinical identification. At present, except for the 16S rRNA gene sequencing method, there is no molecular biological method for the detection and identification of human Ochrobactrum at home and abroad. Therefore, there is an urgent need to develop a technical method with high sensitivity, strong specificity, and convenient, simple and rapid clinical operation for the detection and identification of Ochrobacterium hominis. Summary of the invention

[0005] The present invention is based on the fluorescent quantitative PCR technology, selects three characteristic genes in the genomic DNA nucleotide sequence of Ochrobacterium and Ochrobacterium hominis, designs primers and probes for the gene sequence, and applies the fluorescent quantitative PCR method to accurately detect and identify the nucleic acid DNA of Ochrobacterium and Ochrobacterium hominis strains. For tissue specimens such as blood, the detection method of the present invention is based on the simultaneous detection of three genes, which greatly improves the sensitivity, specificity and positive detection rate of the detection, and has important reference value for clinical diagnosis.

[0006] In order to provide a method that can effectively distinguish Ochrobacterium hominis from its similar species, the present invention downloads the whole genome nucleotide sequence of Ochrobacterium hominis species, Ochrobacterium hominis species, and strains similar to Ochrobacterium hominis species from the NCBI website, and compares and analyzes the genomic DNA nucleotide sequences of all strains. Through the above analysis, the present invention obtains three specific gene sequence sites, which can effectively distinguish the sequences of Ochrobacter hominis from other similar microorganisms. The present invention designs three sets of primers and probe sequences for the above three sites, and the probe sequences are named probe 16s-probe, probe Osp-probe and probe Hyp-probe, respectively.

[0007] The nucleotide sequence for 16s-probe is located at sites 1540097-1540122 and 1802320-1802345 of the reverse-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 1 (GenBank: CP000758.1); and sites 1602609-1602634 of the forward-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 2 (GenBank: CP000759.1). The protein encoded by the 16s-rRNA gene exists in three copies of the gene in the Ochrobactrum genome.

[0008] The nucleotide sequence of the probe Osp-probe is located at the 57737-57788 position of the reverse-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 1 (GenBank: CP000758.1). The protein encoded by the probe Osp-probe is a surface antigen protein. There is one copy of the gene in the genome of Ochrobactrum anthropi.

[0009] The nucleotide sequence of the probe Hyp-probe is located at sites 607436-607484 of the positive-strand DNA and sites 843602-843630 of the reverse-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 1 (GenBank: CP000758.1), and sites 1084351-1084379 of the positive-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 2 (GenBank: CP000759.1). The protein it encodes is a hypothetical protein, and there are three copies of this gene in the Ochrobactrum genome.

[0010] Therefore, in a first aspect, the present invention provides the use of the above-mentioned site in identifying Ochrobacterium hominis.

[0011] The present invention also designs corresponding probe and primer combinations for the above-mentioned specific sites. Therefore, in a second aspect, the present invention provides a primer and probe combination for identifying Ochrobacterium hominis.

[0012] The third aspect of the present invention provides the use of the primer and probe combination for identifying Ochrobacterium hominis described in the second aspect in the preparation of a product for identifying Ochrobacterium hominis.

[0013] In a fourth aspect, the present invention provides a kit for identifying Ochrobacterium hominis, wherein the kit comprises a primer and probe combination for detecting Ochrobacterium hominis.

[0014] The beneficial effect of one or more of the above technical solutions is that the 16SrRNA gene sequencing method is widely used in the identification of microorganisms, especially pathogenic pathogens, but the result needs to be amplified by ordinary PCR, and the amplified product needs to be sequenced and compared before the identification result can be obtained. Similarly, the sequencing of the 16SrRNA gene of the strain of Ochrobacterium has also been reported, but the direct detection and identification of nucleic acid DNA of Ochrobacterium and Ochrobacterium species strains using the fluorescent quantitative PCR method has not been reported so far. The present invention obtains for the first time a fluorescent quantitative PCR method for detecting and identifying nucleic acid DNA of Ochrobacterium and Ochrobacterium species, and adopts a one-step fluorescent quantitative PCR detection method with dual fluorescent labeling by using three genes for simultaneous detection and probes designed for Ochrobacterium and Ochrobacterium species strains. The simultaneous detection of three specific genes greatly increases the positive rate of the test results and avoids false negative results caused by the loss of the nucleotide sequence of the gene during the extraction of nucleic acid DNA; double labeling can simultaneously analyze the nucleic acid DNA of strains of the genus Ochrobacter and the species of Ochrobacter; one-step detection, that is, in one reaction system, completes the results of two detections and identifications, including the detection and identification of strains of the genus Ochrobacter and the species of Ochrobacter. The method of using the established fluorescent quantitative PCR to detect nucleic acid DNA in pure bacteria or body fluid specimens such as blood has the characteristics of strong specificity. Except for the nucleic acid DNA of the genus Ochrobacter and the species of Ochrobacter, other bacterial species have no specific amplification curves; it has high sensitivity and can be detected as long as there is 1 nucleic acid DNA copy number. In addition, the above-mentioned nucleic acid DNA can be either nucleic acid DNA extracted from pure bacteria or bacterial nucleic acid DNA extracted from blood or other body fluids. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 The fluorescence quantitative PCR detection sensitivity described in Example 1;

[0017] Among them, the amount of DNA was 3.67 fg, and the Ct value was 35.38;

[0018] Figure 2 The fluorescence curve of DNA of nucleic acid of strains of Ochrobacterium genus in 32 clinical blood specimens was detected by using primers and probes of Ochrobacterium genus in Example 1;

[0019] Among them, the curve numbers 1-8 in the figure are 8 Ochrobacterium-positive specimens;

[0020] Figure 3 The fluorescence curve of DNA of nucleic acid of Ochrobacterium species strains in 32 blood specimens was detected by using primers and probes of Ochrobacterium species in Example 1;

[0021] The curves marked with numbers 1-6 in the figure are the positive sample numbers, among which 1, 2 and 3 are the positive results of the detection of probes Osp-Probe and Hyp-Probe, and curves 4, 5 and 6 are the positive curve results of the detection of only one probe among the probes Osp-Probe and Hyp-Probe. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0023] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0024] As introduced in the background technology, Ochrobacterium hominis is a pathogen that causes human disease. Since Ochrobacterium hominis has a high similarity in the nucleotide sequence of nucleic acid DNA of microorganisms of similar species, it is easy to cause misdetection. In order to solve the above technical problems, the present invention proposes a method for identifying Ochrobacterium hominis, which simultaneously detects and identifies the nucleic acid DNA of Ochrobacterium species and strains through three specific gene sites, dual fluorescent labeling, and a one-step method, greatly improving the positive rate of detection.

[0025] In a first aspect, the present invention provides the use of any of the following sites in identifying Ochrobactrum hominis, the sites comprising:

[0026] (1) Ochrobactrum anthropi ATCC 49188 chromosome 1 reverse strand DNA at positions 1540097-1540122 and positions 1802320-1802345, and Ochrobactrum anthropi ATCC 49188 chromosome 2 forward strand DNA at positions 1602609-1602634;

[0027] (2) Ochrobactrum anthropi ATCC 49188 chromosome 1 reverse strand DNA at positions 57737-57788;

[0028] (3) Ochrobactrum anthropi ATCC 49188 chromosome 1 positive strand DNA at positions 607436-607484 and reverse strand DNA at positions 843602-843630, and Ochrobactrum anthropi ATCC 49188 chromosome 2 positive strand DNA at positions 1084351-1084379.

[0029] It has been verified by the present invention that the above-mentioned specific nucleotide sequence can be used to identify Ochrobacterium hominis from other similar species, and the specific development direction of the application includes the development of detection methods and the development of detection products. The detection method includes but is not limited to identifying the sequence containing the above-mentioned sites by means of PCR method, immunoblotting, enzyme-linked immunosorbent assay, biological sequencing, etc. In one embodiment provided by the present invention, PCR amplification is used to identify the above-mentioned three sites together, and the primers are primer combinations of the above-mentioned three sites.

[0030] Furthermore, the sequence number of the above-mentioned Ochrobactrum anthropi ATCC 49188 chromosome 1 is GenBank: CP000758.1; the sequence number of the above-mentioned Ochrobactrum anthropi ATCC 49188 chromosome 2 is GenBank: CP000759.1.

[0031] Among the above three sites, the site (1) is mainly for the detection and identification of strains of the genus Ochrobacter, and can be used to identify similar bacterial species including but not limited to Leptospira, Yersinia enterocolitica (0:9), Vibrio cholerae, Escherichia coli (O:157), Mycobacterium tuberculosis, Bacillus anthracis, Salmonella typhi, etc.; the above sites (2) and (3) are for the detection and identification of strains within the species of Ochrobacter hominis.

[0032] In a second aspect of the present invention, a primer and probe combination for identifying Ochrobacterium hominis is provided, wherein the sequences of the probe and primer are as follows:

[0033] Forward primer 16s-up: 5′-GTCCAGCCGAACTGAAAGACAC-3′ (SEQ ID NO. 1);

[0034] Reverse primer 16s-down: 5'-CGTAAACGATGAATGTTAGCCGTT-3' (SEQ ID NO. 2);

[0035] Probe 16s-probe: 5'-ATCTCTGTGTCCGCGACCGGTATGTC-3' (SEQ ID NO. 3);

[0036] Forward primer Osp-up: 5'-TCTTCCTTCTTGTCCTCGCCC-3' (SEQ ID NO. 4);

[0037] Reverse primer Osp-down: 5'-CGGCTCAATTTTCCTCTTTCATC-3' (SEQ ID NO. 5);

[0038] Probe Osp-probe: 5'-ACAGACGAACACCAAAGATCTCGAACGCCAAA-3' (SEQ ID NO. 6);

[0039] Forward primer Hyp-up: 5'-GCGAGCAGCACGCAGCTTC-3' (SEQ ID NO. 7);

[0040] Reverse primer Hyp-down: 5'-GTCGCATTTATTCGGCCTCATAC-3' (SEQ ID NO. 8);

[0041] Probe Hyp-probe: 5'-ACGCCATCTCAAAAGCACTTGGCGATATC-3' (SEQ ID NO. 9).

[0042] Preferably, the above primer and probe combination is suitable for fluorescent PCR detection method, and the probe is modified with a fluorescent group and a quenching group; considering that the identification ranges of the above three sites are different, in one embodiment verified by the present invention, the 5' end of the probe 16s-probe is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ-1; the 5' end of the probe Osp-probe and the probe Hyp-probe are labeled with a fluorescent group Cy5, and the 3' end is labeled with a quenching group BHQ-2. For the above fluorescent labeling method, the positive FAM fluorescent labeling (wavelength 495nm-520nm) indicates that the nucleic acid DNA of the strain of the genus Ochrobacter is present in the specimen; the positive Cy5 fluorescent labeling (wavelength 646nm-662nm) indicates that the nucleic acid DNA of the strain of the species Ochrobacter hominis is present in the specimen.

[0043] The third aspect of the present invention provides the use of the primer and probe combination for identifying Ochrobacterium hominis described in the second aspect in the preparation of a product for identifying Ochrobacterium hominis.

[0044] In the third aspect, the product for identifying Ochrobactrum hominis is one of, but not limited to, a detection kit, a detection platform, a detection system or a detection device. The detection kit is preferably a kit for detection based on a fluorescent PCR method.

[0045] In a fourth aspect, the present invention provides a kit for identifying Ochrobacterium hominis, the kit comprising a primer and probe combination for detecting Ochrobacterium hominis, the sequence of the primer and probe combination being shown in SEQ ID NO.1-9.

[0046] Preferably, the kit also includes a reagent for extracting nucleic acid from a specimen; the specimen is one or more of pure bacteria, serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, etc. Further, the specimen is a serum, plasma or whole blood specimen.

[0047] Preferably, the kit further comprises a PCR reaction system, and the PCR reaction system comprises at least a PCR enzyme complex and a buffer reagent.

[0048] Preferably, the detection kit is used as follows: using the nucleic acid extracted from the specimen as a template, and performing PCR amplification through a fluorescent quantitative PCR reaction using the primers and probes shown in SEQ ID NO: 1-9; further, the conditions of the fluorescent quantitative PCR reaction are: pre-denaturation at 90-96°C for 2-4 min; denaturation at 90-96°C for 4-6 sec, annealing at 55-65°C for 15-25 sec, 35-45 cycles, and finally extension at 35-45°C for 25-35 sec; collecting fluorescence signals during annealing.

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0050] Example 1

[0051] This embodiment provides a method for detecting nucleic acid DNA of Ochrobacterium genus and Ochrobacterium hominis strains in a specimen by using a fluorescent quantitative PCR method:

[0052] The nucleotide sequences of Ochrobacterium hominis, Ochrobacterium hominis, and strains close to Ochrobacterium hominis were downloaded from the NCBI website. The genomic DNA nucleotide sequences of all strains were compared and analyzed. The DNAstar analysis software was used to design primers and probes specific for identifying Ochrobacterium and Ochrobacterium hominis strains. Fluorescence quantitative PCR technology was used to quickly and accurately detect and identify the nucleic acid DNA of Ochrobacterium and Ochrobacterium hominis strains. After analysis, three specific gene sequences were obtained, and three sets of primers and probe sequences were designed. The probe sequences were named probe 16s-probe, probe Osp-probe, and probe Hyp-probe. The nucleotide sequence for 16s-probe is located at the 1540097-1540122 site and the 1802320-1802345 site of the reverse-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 1 (GenBank: CP000758.1); and the 1602609-1602634 site of the forward-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 2 (GenBank: CP000759.1). The protein encoded by the 16s-rRNA gene has three copies in the human Ochrobactrum genome. The nucleotide sequence for the probe Osp-probe is located at the 57737-57788 site of the reverse-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 1 (GenBank: CP000758.1). The protein encoded by the 16s-rRNA gene is the surface antigen protein. The nucleotide sequence of the probe Hyp-probe is located at the 607436-607484 site of the positive-strand DNA and the 843602-843630 site of the reverse-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 1 (GenBank: CP000758.1), and the 1084351-1084379 site of the positive-strand DNA of Ochrobactrum anthropi ATCC 49188 chromosome 2 (GenBank: CP000759.1). The protein encoded by it is a hypothetical protein with unknown function. There are 3 copies of this gene in the genome of Ochrobactrum anthropi. The specific primer and probe nucleotide sequences are as follows:

[0053] Forward primer 16s-up: 5′-GTCCAGCCGAACTGAAAGACAC-3′ (SEQ ID NO. 1);

[0054] Reverse primer 16s-down: 5'-CGTAAACGATGAATGTTAGCCGTT-3' (SEQ ID NO. 2);

[0055] Probe 16s-probe: 5'-ATCTCTGTGTCCGCGACCGGTATGTC-3' (SEQ ID NO. 3);

[0056] Forward primer Osp-up: 5'-TCTTCCTTCTTGTCCTCGCCC-3' (SEQ ID NO. 4);

[0057] Reverse primer Osp-down: 5'-CGGCTCAATTTTCCTCTTTCATC-3' (SEQ ID NO. 5);

[0058] Probe Osp-probe: 5'-ACAGACGAACACCAAAGATCTCGAACGCCAAA-3' (SEQ ID NO. 6);

[0059] Forward primer Hyp-up: 5'-GCGAGCAGCACGCAGCTTC-3' (SEQ ID NO. 7);

[0060] Reverse primer Hyp-down: 5'-GTCGCATTTATTCGGCCTCATAC-3' (SEQ ID NO. 8);

[0061] Probe Hyp-probe: 5'-ACGCCATCTCAAAAGCACTTGGCGATATC-3' (SEQ ID NO. 9).

[0062] Probe sequence: The 5' end of the probe 16s-probe is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ-1; the 5' end of the probe Osp-probe and the probe Hyp-probe are labeled with the fluorescent group Cy5, and the 3' end is labeled with the quenching group BHQ-2.

[0063] The primers and probes include: forward primer 16s-up, reverse primer 16s-down and probe 16s-probe, which are used for the detection and identification of strains of the genus Ochrobacter; forward primers Osp-up and Hyp-up, reverse primers Osp-down and Hyp-down, probes Osp-probe and Hyp-probe, which are used for the detection and identification of strains within the species of Ochrobacter.

[0064] 1. Primer specificity verification

[0065] 1) Fluorescence quantitative PCR amplification, forward primers 16s-up, Osp-up and Hyp-up; reverse primers 16s-up, Osp-down and Hyp-down; probes 16s-probe, Osp-probe and Hyp-probe, fluorescent quantitative PCR was used to amplify the nucleic acid DNA of 22 strains of 19 biotypes of 6 species of Brucella, as well as the nucleic acid DNA of bacteria such as Leptospira, Yersinia enterocolitica (0:9), Vibrio cholerae, Escherichia coli (O:157), Mycobacterium tuberculosis, Bacillus anthracis and Salmonella typhi.

[0066] 2) The fluorescence quantitative PCR reaction system includes: 10 μL of fluorescence quantitative PCR enzyme complex, 0.3 μL of forward primer 16s-up, Osp-up and Hyp-up solution, 0.3 μL of reverse primer 16s-dpwn, Osp-down and Hyp-down solution, 0.4 μL of probe 16s-probe, Osp-probe and Hyp-probe solution, 2 μL of bacterial nucleic acid DNA in step 1) as template, and double distilled water is added to the volume of 20 μL.

[0067] 3) Fluorescence quantitative PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 sec, annealing at 60°C for 20 sec, 40 cycles, and final extension at 40°C for 30 sec; fluorescence signals were collected during annealing.

[0068] 4) Preferably, in step 2), the concentration of the forward primer, reverse primer and probe solution is 10 μM.

[0069] 5) Optionally, in step 2), the fluorescent quantitative PCR enzyme complex is 2×Premix Ex Taq TM (TaKaRa,RR390A).

[0070] 2. Primer sensitivity verification

[0071] 1) Fluorescence quantitative PCR amplification, forward primers 16s-up, Osp-up and Hyp-up; reverse primers 16s-up, Osp-down and Hyp-down; probes 16s-probe, Osp-probe and Hyp-probe, nucleic acid DNA template is 2 μL of nucleic acid DNA extracted from pure bacteria or blood specimens;

[0072] 2) The fluorescence quantitative PCR reaction system includes: 10 μL of fluorescence quantitative PCR enzyme complex, 0.3 μL of forward primer 16s-up, Osp-up and Hyp-up solution, 0.3 μL of reverse primer 16s-dpwn, Osp-down and Hyp-down solution, 0.4 μL of probe 16s-probe, Osp-probe and Hyp-probe solution, 2 μL of extracted nucleic acid DNA as template, and double distilled water added to 20 μL volume. The concentration of forward primer, reverse primer and probe solution is 10 μM; the fluorescence quantitative PCR enzyme complex is 2×Premix Ex Taq TM (TaKaRa,RR390A).

[0073] 3) Fluorescence quantitative PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 sec, annealing at 60°C for 20 sec, 40 cycles; final extension at 40°C for 30 sec; fluorescence signals were collected during annealing.

[0074] 3. Results

[0075] 1) Primer specificity: The primers and probes designed for the fluorescent quantitative PCR of the present invention have high specificity, and no fluorescence curve (no Ct value) is amplified for nucleic acid DNA of non-Ochrobacter strains. After submitting the designed primers and probes to the NCBI website for comparison and analysis, it was found that there was no complete match with the nucleotide sequence of nucleic acid DNA of other non-Ochrobacter strains, which conforms to the probe design principle of fluorescent quantitative PCR.

[0076] 2) Primer sensitivity: The fluorescent quantitative PCR method designed in the present invention has high sensitivity and can detect 3.67 fg of DNA. The Ct value is 35.38, that is, the PCR reaction system can detect as long as there is 1 nucleic acid DNA copy number. Figure 1 .

[0077] 3) Using commercial blood and other specimen extraction kits, bacterial nucleic acid DNA was extracted from clinical blood specimens, and the Ochrobacterium humanum detection and identification kit was used for detection. The results are as follows:

[0078] The present invention randomly selects 32 patients who visit the brucellosis clinic, collects 32 blood specimens from them, extracts nucleic acid DNA by using a bacterial genome DNA extraction kit for blood specimens, and uses the kit for detection. Figure 2 There were 8 specimens positive for Ochrobacterium nucleic acid DNA; Figure 3 There were 6 samples that were positive for Ochrobacterium hominis nucleic acid DNA, and another 2 samples that were positive for Ochrobacterium genus nucleic acid DNA, but negative for Ochrobacter hominis nucleic acid DNA. The specific identification results are shown in Table 1 below:

[0079] Table 1 Comparison of the test results of 32 clinical blood specimens by VITEK 2 bacterial identification system and Ochrobacterium hominis kit

[0080]

[0081] As can be seen from the results in the above table, the identification results of 8 cases of Ochrobacterium provided by the present invention are consistent with the identification results of the VITEK 2 bacterial identification system, and 6 of them are positive for Ochrobacterium hominis infection, and the accuracy of the detection kit provided by the present invention reaches 100%. It is confirmed that the method of the present invention can effectively distinguish Ochrobacter hominis and its similar strains, and its application in clinical detection is helpful to provide a more accurate clinical diagnosis basis.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. SEQUENCE LISTING <110> Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention <120> A primer, probe combination and detection kit for identifying Ochrobacterium hominis <130> 2010 <160> 9 <170> PatentIn version 3.3 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <400> 1 gtccagccga actgaaagac ac 22 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <400> 2 cgtaaacgat gaatgttagc cgtt 24 <210> 3 <211> 26 <212> DNA <213> Artificial sequence <400> 3 atctctgtgt ccgcgaccgg tatgtc 26 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <400> 4 tcttccttct tgtcctcgcc c 21 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> 5 cggctcaatt ttcctctttc atc 23 <210> 6 <211> 32 <212> DNA <213> Artificial sequence <400> 6 acagacgaac accaaagatc tcgaacgcca aa 32 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <400> 7 gcgagcagca cgcagcttc 19 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <400> 8 gtcgcattta ttcggcctca tac 23 <210> 9 <211> 29 <212> DNA <213> Artificial sequence <400> 9 acgccatctc aaaagcactt ggcgatatc 29

Claims

1. A primer and probe combination for identifying Ochrobacterium hominis, characterized in that: The probe and primer sequences are as follows: Forward primer 16s-up: 5′-GTCCAGCCGAACTGAAAGACAC-3′; Reverse primer 16s-down: 5′-CGTAAACGATGAATGTTAGCCGTT -3′; Probe 16s-probe: 5′-ATCTCTGTGTCCGCGACCGGTATGTC -3′; Forward primer Osp-up: 5′-TCTTCCTTCTTGTCCTCGCCC-3′; Reverse primer Osp-down: 5′-CGGCTCAATTTTCCTCTTTCATC -3′; Osp-probe: 5′-ACAGACGAACACCAAAGATCTCGAACGCCAAA -3′; Forward primer Hyp-up: 5′-GCGAGCAGCACGCAGCTTC-3′; Reverse primer Hyp-down: 5′-GTCGCATTTATTCGGCCTCATAC -3′; Hyp-probe: 5′-ACGCCATCTCAAAAGCACTTGGCGATATC -3′.

2. The primer and probe combination for identifying Ochrobacterium hominis according to claim 1, characterized in that: The primer and probe combination is suitable for a fluorescent PCR detection method, and the probe is modified with a fluorescent group and a quenching group.

3. The primer and probe combination for identifying Ochrobacterium hominis according to claim 1, characterized in that: The 5' end of the probe 16s-probe is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ-1; the 5' ends of the probe Osp-probe and the probe Hyp-probe are labeled with a fluorescent group Cy5, and the 3' ends are labeled with a quenching group BHQ-2.

4. Use of the primer and probe combination according to claim 1 or 2 in the preparation of a product for identifying Ochrobacterium hominis.

5. The use of the primer and probe combination as claimed in claim 4 in the preparation of a product for identifying Ochrobacterium hominis, characterized in that: The product for identifying Ochrobactrum hominis is a kit for detection based on a fluorescent PCR method.

6. A kit for identifying Ochrobactrum hominis, characterized in that: The kit includes a primer and probe combination for detecting Ochrobacterium hominis, and the sequences of the primer and probe combination are shown in SEQ ID NO.1-9.

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