Kit and method for identifying multiple fluorescent quantitative PCR (Polymerase Chain Reaction) of fusobacterium
By designing a multi-fluorescence quantitative PCR kit with specific primers and probes, the problem of multiple fluorescence PCR identification in the prior art is solved, and accurate detection of multiple Fusobacter species and subspecies is achieved, which improves detection efficiency and accuracy.
Patent Information
- Application Number
- CN202510195045.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-04-18
AI Technical Summary
It is difficult for the prior art to achieve multiple fluorescence PCR identification of similar species, and there are problems such as false positives, false negatives, and low amplification efficiency.
Multiple fluorescence quantitative PCR kits and methods for identifying Florabacterium were designed. By screening out specific genetic markers and designing highly specific primers and probes, the binding efficiency of primers and probes is improved, and cross-reactions and the formation of primer dimers are avoided.
Simultaneous detection of multiple target bacteria is achieved, the accuracy and efficiency of detection is improved, and the probability of false positives and false negatives is reduced.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of microbial identification and detection, and in particular, to a multiplex fluorescence quantitative PCR kit and method for identifying Fusobacterium. Background Art
[0002] Fusobacterium is a common Gram-negative, non-spore-forming anaerobic bacillus in clinical practice. As a conditional pathogen, it is associated with a variety of human diseases, such as adverse pregnancy diseases (chorioamnionitis, neonatal sepsis, preeclampsia), gastrointestinal diseases (colorectal cancer, inflammatory bowel disease, appendicitis), cardiovascular disease, rheumatoid arthritis, respiratory tract infection, Lemierre syndrome and Alzheimer's disease, etc. More and more studies have shown that Fusobacterium is common in colorectal cancer, especially Fusobacterium nucleatum and its subspecies are related to the carcinogenic mechanism of colorectal cancer. However, there is no consistent conclusion on the statistical results of the prevalence of different species of Fusobacterium in human diseases such as colorectal cancer, which seriously hinders its population epidemiological analysis and clinical diagnosis application. Therefore, identifying the species level of intestinal Fusobacterium community is of great significance for exploring the relationship between Fusobacterium members and human diseases such as colorectal cancer, and can provide a technical basis for the early diagnosis of Fusobacterium-related diseases. However, there is currently a lack of an accurate and efficient detection method that can be used to simultaneously identify common species of Fusobacterium and subtypes of Fusobacterium nucleatum. Different species of Fusobacterium and different subspecies of Fusobacterium nucleatum have high similarity in gene sequences, but they differ in their biological characteristics, colonization and pathogenicity. Therefore, accurate identification of Fusobacterium species / subspecies is also of far-reaching significance for understanding its pathogenic mechanism.
[0003] Different from the single-plex PCR technology that can only detect one target at a time, the multiplex fluorescence PCR technology can detect multiple targets at the same time. Its basic principle is to add more than two pairs of primers and probes to the same PCR reaction system, so as to simultaneously perform PCR amplification on two or more different nucleic acid fragments. Its advantage is that it can solve the situation where multiple targets need to be amplified multiple times, which not only saves time but also saves the cost of consumables. However, the reaction system of multiplex fluorescence PCR may increase the probability of false positives and false negatives, and may also have problems such as low amplification efficiency and uneven amplification efficiency. Therefore, the challenges that may be faced when designing the reaction system include difficulties in primer design, cross-reactions, inconsistent annealing temperatures, template competition, differences in amplification efficiency, detection method limitations, inhibitory factors, etc. Summary of the invention
[0004] In view of the shortcomings of the existing methods, the present application proposes a kit and method for multiplex fluorescence quantitative PCR for identifying Fusobacterium, so as to solve the technical problem that it is difficult to perform multiplex fluorescence PCR on similar species in the related technology.
[0005] In a first aspect, the present application provides a multiplex fluorescence quantitative PCR kit for identifying Fusobacterium, comprising: Reaction solution A, each group of the reaction solution A includes at least one specific primer for a target bacterium, a specific probe, an internal standard and a buffer; Reaction solution B, including hot-start Taq antibody enzyme, UDG enzyme and dNTPs; Standard solutions, including host cell nucleic acids; Controls include plasmid and host cell nucleic acids containing fragments of all target bacteria.
[0006] Optionally, each group of the reaction solution A includes 1 to 3 specific primers and specific probes for target bacteria; the target bacteria that can be detected by the same group of the reaction solution A are of different genera, species or subspecies; and 1 to 3 groups of the reaction solution A are set.
[0007] Further optionally, the target bacteria and its specific primers and specific probes include at least one of the following: Fusobacterium death, Fusobacterium deadly , the specific primer pair includes Fm-F and Fm-R, the sequences of which are shown in SEQ No.1 and SEQ No.2, respectively, and the specific probe includes Fm-P, the sequence of which is shown in SEQ No.3; Fusobacterium mutans, Fusobacterium varius , the specific primer pair includes Fv-F and Fv-R, the sequences of which are shown in SEQ No.4 and SEQ No.5, respectively, and the specific probe includes Fv-P, the sequence of which is shown in SEQ No.6; Fusobacterium ulcerans, Fusobacterium ulcerans , the specific primer pair includes Fu-F and Fu-R, the sequences of which are shown in SEQ No.7 and SEQ No.8 respectively, and the specific probe includes Fu-R, the sequence of which is shown in SEQ No.9; Fusobacterium necroticum, Fusobacterium necrophorum , the specific primer pair includes Fne-F and Fne-R, the sequences of which are shown in SEQ No.10 and SEQ No.11 respectively, and the specific probe includes Fne-P, the sequence of which is shown in SEQ No.12; Fusobacterium nucleatum subsp. nucleatum, Fusobacterium nucleatum subsp. nucleatum , the specific primer pair includes Fnn-F and Fnn-R, the sequences of which are shown in SEQ No.13 and SEQ No.14, respectively, and the specific probe includes Fnn-P, the sequence of which is shown in SEQ No.15; Fusobacterium nucleatum subsp. Fusobacterium nucleatum subsp. animalis, the specific primer pair includes Fna-F and Fna-R, the sequences of which are shown in SEQ No.16 and SEQ No.17, respectively, and the specific probe includes Fna-P, the sequence of which is shown in SEQ No.18; Fusobacterium nucleatum subsp. wenkelii, Fusobacterium nucleatum subsp. vincentii , the specific primer pair includes Fnv-F and Fnv-R, the sequences of which are shown in SEQ No.19 and SEQ No.20 respectively, and the specific probe includes Fnv-P, the sequence of which is shown in SEQ No.21; Fusobacterium nucleatum subsp. polymorphum, Fusobacterium nucleatum subsp. polymorphic The specific primer pair includes Fnp-F and Fnp-R, whose sequences are shown in SEQ No.22 and SEQ No.23 respectively, and the specific probe includes Fnp-P, whose sequence is shown in SEQ No.24.
[0008] Furthermore, the reaction solution A comprises: The first reaction solution A comprises specific primers and specific probes of Fusobacterium nucleatum subspecies polymorphum, Fusobacterium nucleatum subspecies nucleatum and Fusobacterium nucleatum subspecies wenkelii; The second reaction solution A includes specific primers and specific probes for Fusobacterium necroticum, Fusobacterium nucleatum subspecies animalum, and Fusobacterium mortis; The third reaction solution A includes specific primers and specific probes for Fusobacterium variatum and Fusobacterium ulcerans.
[0009] Alternatively, the internal standard includes glyceraldehyde-3-phosphate dehydrogenase, the specific primer pair includes GAPDH-F and GAPDH-R, the sequences of which are shown in SEQ No. 25 and SEQ No. 26 respectively, and the specific probe includes GAPDH-P, the sequence of which is shown in SEQ No. 27.
[0010] Further optionally, the fluorescent label on the specific probe includes at least one of 6-carboxyfluorescein, sulforhodamine, cyanine dye Cy5, and a dye dedicated to PCR equipment.
[0011] Alternatively, the host cell nucleic acid comprises human cell nucleic acid.
[0012] In a second aspect, the present application also provides a method for multiplex fluorescence quantitative PCR for identifying Fusobacterium, using the multiplex fluorescence quantitative PCR kit for identifying Fusobacterium as described above, which comprises the following steps: Obtaining a sample and extracting total DNA from the sample; The total DNA of each sample is mixed with each group of reaction solution A, and each group of reaction solution A is also mixed with reaction solution B and buffer to form a reaction system of a specified volume; Placing the reaction system, standard solution, and reference substance on a PCR amplification instrument for amplification; The detection of target bacteria in the sample is confirmed based on the color development of the specific probe.
[0013] Optionally, the sample is from at least one of feces, tissue, blood and body fluid; the tissue comprises at least one of tumor tissue, non-tumor pathological tissue and healthy tissue.
[0014] Alternatively, the total DNA concentration of the sample mixed with each set of reaction solution A is 1×10 3 ~1×10 7 copies / mL range.
[0015] Furthermore, each reaction system containing the total DNA of the sample is amplified simultaneously, and the amplification conditions are consistent.
[0016] Furthermore, the standard solution and the control substance present a specified result after amplification, which confirms that the current round of amplification is valid.
[0017] The beneficial technical effects brought about by the technical solution provided by the embodiment of the present application include: The present application utilizes the screened specific genetic markers and designs highly specific primers based on the specific genetic markers, thereby improving the binding efficiency of primers and probes, avoiding the cross-reaction of primers and the formation of primer dimers, and through the reasonable combination of different target bacteria, the present application can achieve simultaneous detection of multiple targets.
[0018] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. DETAILED DESCRIPTION
[0019] The following is an embodiment of the present application described in conjunction with the present application. It should be understood that the implementation methods described below are exemplary descriptions for explaining the technical solutions of the embodiments of the present application and do not constitute a limitation on the technical solutions of the embodiments of the present application.
[0020] Those skilled in the art will appreciate that, unless expressly stated, the "said" and "the" used herein may also include plural forms. It should be further understood that the term "including" used in the specification of the present application refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the implementation of other features, information, data, steps, operations, elements, components and / or combinations thereof supported by the technical field. The term "and / or" used herein refers to at least one of the items defined by the term, for example, "A and / or B" may be implemented as "A", or as "B", or as "A and B".
[0021] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below.
[0022] The existing classification and identification technologies for Fusobacterium in tissues or feces mainly include: (1) Bacterial staining and analysis with ordinary optical microscope or electron microscope: Staining and ordinary optical microscope can provide a simple understanding of the structure and morphological characteristics of bacteria. However, due to the small morphological differences of bacteria, simple optical microscopes cannot complete bacterial species identification. Although electron microscopes have high resolution, they still cannot identify bacterial species. In addition, the maintenance cost and time cost are high, and the operation is difficult. (2) Traditional microbial identification and community analysis: Most of them use the method of pure strain identification after strain isolation and culture. The isolation and culture is very difficult, time-consuming, and inefficient, and is not suitable for batch sample testing; (3) Molecular biology techniques, such as PCR technology, 16s rDNA sequencing technology, metagenomic sequencing analysis, and digital PCR. Singleplex PCR technology is widely used because of its simple operation, but it can only detect one target at a time. When performing multi-target detection, it is time-consuming and labor-intensive. At present, there is only singleplex PCR detection technology for Fusobacterium nucleatum on the market. Other molecular detection technologies are expensive, time-consuming, and are greatly affected by complex factors in tumors and feces, so they cannot be specifically classified.
[0023] The present invention screens specific genetic markers through large-scale genomes, and designs highly specific primers based on the markers, specifically, designs and verifies specific primers and probes for four types (subspecies) of Fusobacterium nucleatum and four non-Fusobacterium nucleatum (also Fusobacterium genus), as well as endogenous internal standard specific primers and probes. The specific bacterial species are: Fusobacterium mortis ( Fusobacterium mortiferum, Fm. ), Fusobacterium necroticum ( Fusobacterium necrophorum, End ), Fusobacterium ulcerans ( Fusobacterium ulcerans, Fu ), Fusobacterium mutans ( Fusobacterium varius, Fv ), and Fusobacterium nucleatum subsp. nucleatum ( Fusobacterium nucleatum subsp.nucleatum, Fnn ), Fusobacterium nucleatum subsp. animalium ( Fusobacterium nucleatum subsp. animalis, Fna )、Fusobacterium nucleatum subsp. wenkelii( Fusobacterium nucleatum subsp. vincentii, Fnv ) and Fusobacterium nucleatum subsp. polymorpha ( Fusobacterium nucleatum subsp. polymorph, Fnp ). The design of the primers and probes is based on an in-depth analysis of the gene sequences of Fusobacterium nucleatum and its related strains, ensuring that each primer probe can specifically bind to a specific gene region of the target strain, thereby achieving accurate detection. The specific primer and probe sequences are shown in Table 1 below: Table 1 Primer and probe sequences for multiplex PCR detection of Fusobacterium
[0024] Further, the multiplex fluorescence quantitative PCR system formed by the specific primer pairs and specific probes in the above table was verified and optimized. The verification content includes the performance index evaluation of the optimal concentration, specificity, sensitivity and amplification efficiency of the primers and probes to ensure their accuracy and reliability in the multiplex fluorescence PCR detection system. The optimal concentration, specificity, sensitivity and amplification efficiency of the primers and probes are shown in the results below.
[0025] In one possible embodiment, the specific primers and specific probes designed above for the eight target bacteria (targets), as well as the specific primers and specific probes for the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are used to form a kit that can be used in a process-based manner. The kit includes reaction solution A, reaction solution B, standard solution and reference substance.
[0026] Among them, the reaction solution A is set to 1 to 3 groups, and each group of reaction solution A includes 1 to 3 target specific primers and specific probes, so that the kit can achieve the identification of 1 to 9 targets. In other embodiments, the number of reaction solution A groups and the number of targets included in each group of reaction solution A can also be appropriately expanded to achieve multiplex fluorescence quantitative PCR amplification and identification according to general requirements.
[0027] Referring to Table 2, in this embodiment, 3 groups of reaction liquid A are set, and each group of reaction liquid A includes specific primers and specific probes of 2 or 3 targets, thereby covering the aforementioned 8 target bacteria. After testing, the 8 target bacteria are combined into the same group of reaction liquid A in the following manner: reaction liquid A1 includes specific primers and specific probes of Fusobacterium nucleatum polymorphic subspecies, Fusobacterium nucleatum subspecies nucleatum and Fusobacterium nucleatum wenshi subspecies; reaction liquid A2 includes specific primers and specific probes of Fusobacterium necrotis, Fusobacterium nucleatum animal subspecies and Fusobacterium death; reaction liquid A3 includes specific primers and specific probes of Fusobacterium variable and Fusobacterium ulcerans; the specific specific primers and specific probes used for each target are shown in Table 2. According to the combination of this embodiment, the targets to be detected in the same group of reaction liquid A can be of different genera, different species or different subspecies. When the sequences of specific primers and specific probes are reasonably designed, even two species with very high gene sequence similarity can be identified by multiplex fluorescence quantitative PCR.
[0028] Table 2 Primer and probe combinations and dosages for each target in each reaction tube
[0029] The detection specificity of each reaction solution A was tested at the dosage in Table 2, and the corresponding bacterial solution was used as a sample for testing. The testing process was based on the processing process of general clinical samples: 1 Sample processing and DNA extraction: Use the corresponding nucleic acid extraction kit to extract the total DNA from the sample.
[0030] 2 PCR amplification.
[0031] 2.1 PCR reagent preparation 2.1.1 Take reaction solution A1, reaction solution A2, reaction solution A3, and reaction solution B, thaw at room temperature, shake and mix, and centrifuge for a few seconds before use. Take N×3 PCR reaction tubes and number them as "reaction tube 1", "reaction tube 2", and "reaction tube 3" respectively, where N = number of samples to be tested + standard solution + reference substance.
[0032] Reaction solution B includes hot-start Taq antibody enzyme, UDG enzyme and dNTPs, which are enzymes and synthetic raw materials necessary for fluorescent PCR.
[0033] The samples of this embodiment are mainly derived from human tissue, feces or body fluids, and the detection target is the symbiotic bacteria in the human body. Therefore, the standard solution contains human cell nucleic acid of the human as the host, and the reference substance includes the fragment plasmids of all targets and human cell nucleic acid. The sequences of the fragments of the 8 targets of this embodiment are shown in SEQ No.28 to SEQ No.35. The fragment sequence of the internal standard is shown in SEQ No.36.
[0034] In other embodiments, the enzyme in the reaction solution B may be other polymerases, and the standard solution and reference substance should be adjusted according to the sampling environment and the target.
[0035] The configuration volume of each reaction tube for a single sample is shown in Table 3, and the concentration of the total DNA added to each reaction tube is controlled at 1×10 3 ~1×10 7 The concentration of total DNA can be controlled within the range of 100 copies / mL by concentration or dilution.
[0036] Table 3 Configuration table of Fusobacterium multiplex PCR single sample amplification system
[0037] According to the required total amount, each component was fully mixed and then centrifuged instantly to allow all the liquid on the tube wall to be centrifuged to the bottom of the tube. Then 20µL of the amplification system was dispensed into the PCR reaction tube.
[0038] 2.2 Sample addition 2.2.1 Add 10 μL of standard solution, sample nucleic acid to be tested, and control substance to the above PCR reaction tube, centrifuge for 30 seconds, and place in PCR amplification instrument.
[0039] 2.3 Instrument Setup 2.3.1 This example uses an ABI PCR amplification instrument. Open the "Plate Setup" window, click the Add New Target button, create a new probe, and set the probe detection mode to: Reporter Dye1: FAM, Quencher Dye1: none, Reporter Dye2: VIC, Quencher Dye2: none, Reporter Dye3: Texas red, Quencher Dye3: none, Reporter Dye4: CY5, Quencher Dye4: none; click the "Assign Target Samples" button, set the FBM standard solution (NEG), FBM control and unknown sample (Unknown) in the corresponding order of the samples, and set the sample name in the "Sample Name" column, Passive Reference: none.
[0040] 2.3.2 Open the “Run Method” window and set the cycling conditions as shown in Table 4; and set the PCR reaction volume to 30 µL in “Reaction Volume Per Well”.
[0041] Table 4 Cycling conditions for multiplex PCR amplification of Fusobacterium
[0042] 2.3.3 After the settings are completed, save the file and click the “Start the instrument run” button to run the program.
[0043] 2.3.4 Monitoring process: Switch to the program running page to display the actual situation of the PCR curve gradually growing with the increase of cycles in real time. According to the fluorescent markers connected to each specific probe - FAM (6-carboxyfluorescein), VIC (ABI special dye), Texas Red (sulforhodamine) and CY5 (cyanine dye Cy5), select the corresponding fluorescent channel and only display the changes of the corresponding probe.
[0044] 2.3.5 Save results: After the program ends, it will be automatically saved to the file path.
[0045] 2.4 Results Analysis The results are automatically saved after the reaction is completed. Adjust the Start value, End value and Threshold value of the Baseline according to the analyzed image (users can adjust according to actual conditions. The Start value can be set between 3 and 15, and the End value can be set between 5 and 20. Set the Value of the Threshold in the Log graph window so that the threshold line is in the exponential phase of the amplification curve and the amplification curve of the standard solution is flat or below the threshold line). Click Analysis to automatically obtain the analysis results and read the test results in the "Report" window.
[0046] 2.5 Determination of test results.
[0047] The following requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.
[0048] (1) FBM standard solution: There is no obvious amplification curve in the FAM, VIC, and Texas Red detection channels of FBM reaction tubes 1, 2, and 3, and there is an obvious S-shaped amplification curve in the CY5 detection channel, and the Ct value is ≤35; (2) FBM control: In FBM reaction tubes 1 and 2, the FAM, VIC, and Texras Red detection channels have amplification curves with Ct values ≤ 35, and the CY5 detection channel has or does not have an amplification curve; in FBM reaction tube 3, the FAM and Texras Red detection channels have amplification curves with Ct values ≤ 35, and the CY5 detection channel has or does not have an amplification curve; Further identification criteria for each target are shown in Table 5 below: Table 5 Judgment criteria for Fusobacterium multiplex PCR results
[0049] The reaction solution A is specifically detected using bacterial liquid. The bacterial liquid used should not only include the 8 target bacteria involved in this embodiment, but also non-target bacteria should be used as identification objects to confirm whether false positives or false negatives will occur. The bacterial liquid used as the test sample can be a single bacterial liquid or a mixed bacterial liquid. Referring to the experimental process above, the specific detection results of the reaction system of this application are shown in Table 6. The results in Table 6 show that the specificity of the reaction solution A meets the requirements.
[0050] Table 6 Specificity detection of reaction solution A
[0051] Note: “+” means positive, “-” means negative.
[0052] Furthermore, the sensitivity test of reaction solution A was performed using bacterial solution to confirm the lowest concentration of DNA that can be detected by the reaction system including reaction A and the aforementioned amplification procedure. The results are shown in Table 7. The lower limit of the sample DNA concentration that can be detected in each reaction tube is 1×10 3 The concentration of sample DNA was increased to further test the amplification efficiency of the reaction system including reaction A and the aforementioned amplification procedure. The results are shown in Table 8. The amplification efficiency of each detection target was in the range of 90% to 115%, and the correlation coefficient R 2 ≥0.98, indicating that the amplification efficiency of the reaction system including reaction A and the aforementioned amplification procedure in the present application is qualified.
[0053] Table 7 Sensitivity test results of reaction tubes 1-3
[0054] Table 8 Amplification efficiency test results of reaction tubes 1-3
[0055] After testing the specificity, sensitivity and expansion efficiency, the present application includes a reaction system of reaction A and a matching amplification program, and the relevant reagents can be commercially packaged to form a kit, and instructions for use are attached. The kit of the present application includes reaction solution A, reaction solution B, standard solution and reference substance. Among them, reaction solution A includes specific primers, specific probes, internal standards and buffers for at least one target bacterium. The components and reagent specifications of a single kit can be referred to Table 9.
[0056] Table 9 Fusobacterium multiplex PCR kit components
[0057] In summary, the feasibility of this application has been verified through a series of experiments and positive results have been achieved. In the experimental stage, the amplification efficiency, sensitivity and specificity of this application were comprehensively evaluated to ensure its accuracy and reliability in the field of molecular diagnosis. In addition, in order to further verify the clinical applicability of this application, human tissue samples and stool specimens were used, and the application was externally verified in combination with high-throughput sequencing technology. The sample source can be further amplified to at least one of tumor tissue, non-tumor pathological tissue, healthy tissue, serum, and body fluids (such as cerebrospinal fluid). The experimental results show that this application has demonstrated excellent accuracy and reliability in detecting target biomarkers, providing a new and efficient tool for clinical diagnosis. Through these comprehensive evaluations, we are convinced of the effectiveness of this application in practical applications and have laid a solid foundation for its future clinical application.
[0058] Those skilled in the art will appreciate that the various operations, methods, steps, measures, and schemes in the processes discussed in this application may be alternated, changed, combined, or deleted. Furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and schemes in the related art that are similar to those disclosed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted.
[0059] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "plurality" means two or more.
[0060] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.
[0061] The above is only a partial implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the technical concept of the scheme of the present application, other similar implementation methods based on the technical ideas of the present application are also within the protection scope of the embodiments of the present application.
Claims
1. A multiplex fluorescence quantitative PCR kit for identifying Fusobacterium, characterized in that: It includes: Reaction solution A, each group of the reaction solution A includes at least one specific primer for a target bacterium, a specific probe, an internal standard and a buffer; Reaction solution B, including hot-start Taq antibody enzyme, UDG enzyme and dNTPs; Standard solutions, including host cell nucleic acids; Controls include plasmid and host cell nucleic acids containing fragments of all target bacteria.
2. The multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to claim 1, characterized in that: Each group of the reaction solution A includes 1 to 3 specific primers and specific probes for target bacteria; the target bacteria that can be detected by the same group of the reaction solution A are of different genera, species or subspecies; 1 to 3 groups of the reaction solution A are set.
3. The multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to claim 1, characterized in that: The target bacteria and its specific primers and specific probes include at least one of the following: Fusobacterium death, Fusobacterium mortiferum , the specific primer pair includes Fm-F and Fm-R, the sequences of which are shown in SEQ No.1 and SEQ No.2, respectively, and the specific probe includes Fm-P, the sequence of which is shown in SEQ No.3; Fusobacterium mutans, Fusobacterium varium , the specific primer pair includes Fv-F and Fv-R, the sequences of which are shown in SEQ No.4 and SEQ No.5 respectively, and the specific probe includes Fv-P, the sequence of which is shown in SEQ No.6; Fusobacterium ulcerans, Fusobacterium ulcerans , the specific primer pair includes Fu-F and Fu-R, the sequences of which are shown in SEQ No.7 and SEQ No.8 respectively, and the specific probe includes Fu-R, the sequence of which is shown in SEQ No.9; Fusobacterium necroticum, Fusobacterium necrophorum , the specific primer pair includes Fne-F and Fne-R, the sequences of which are shown in SEQ No.10 and SEQ No.11 respectively, and the specific probe includes Fne-P, the sequence of which is shown in SEQ No.12; Fusobacterium nucleatum subsp. nucleatum, Fusobacterium nucleatum subsp.nucleatum , the specific primer pair includes Fnn-F and Fnn-R, the sequences of which are shown in SEQ No.13 and SEQ No.14, respectively, and the specific probe includes Fnn-P, the sequence of which is shown in SEQ No.15; Fusobacterium nucleatum subsp. Fusobacterium nucleatum subsp. animalis , the specific primer pair includes Fna-F and Fna-R, the sequences of which are shown in SEQ No.16 and SEQ No.17, respectively, and the specific probe includes Fna-P, the sequence of which is shown in SEQ No.18; Fusobacterium nucleatum subsp. wenkelii, Fusobacterium nucleatum subsp. vincentii , the specific primer pair includes Fnv-F and Fnv-R, the sequences of which are shown in SEQ No.19 and SEQ No.20 respectively, and the specific probe includes Fnv-P, the sequence of which is shown in SEQ No.21; Fusobacterium nucleatum subsp. polymorphum, Fusobacterium nucleatum subsp. polymorphum The specific primer pair includes Fnp-F and Fnp-R, whose sequences are shown in SEQ No.22 and SEQ No.23 respectively, and the specific probe includes Fnp-P, whose sequence is shown in SEQ No.
24.
4. The multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to claim 3, characterized in that: The reaction solution A comprises: The first reaction solution A comprises specific primers and specific probes of Fusobacterium nucleatum subspecies polymorphum, Fusobacterium nucleatum subspecies nucleatum and Fusobacterium nucleatum subspecies wenkelii; The second reaction solution A includes specific primers and specific probes for Fusobacterium necroticum, Fusobacterium nucleatum subspecies animalum, and Fusobacterium mortis; The third reaction solution A includes specific primers and specific probes for Fusobacterium variatum and Fusobacterium ulcerans.
5. The multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to claim 1, characterized in that: The internal standard includes glyceraldehyde-3-phosphate dehydrogenase, the specific primer pair includes GAPDH-F and GAPDH-R, the sequences of which are shown in SEQ No. 25 and SEQ No. 26 respectively, and the specific probe includes GAPDH-P, the sequence of which is shown in SEQ No.
27.
6. The multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to claim 1, characterized in that: The fluorescent label on the specific probe includes at least one of 6-carboxyfluorescein, sulforhodamine, cyanine dye Cy5, and a special dye for PCR equipment.
7. The multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to claim 1, wherein the host cell nucleic acid comprises human cell nucleic acid.
8. A method for multiplex fluorescence quantitative PCR for identifying Fusobacterium, using the multiplex fluorescence quantitative PCR kit for identifying Fusobacterium according to any one of claims 1 to 7, characterized in that: It includes the following steps: Obtaining a sample and extracting total DNA from the sample; The total DNA of each sample is mixed with each group of reaction solution A, and each group of reaction solution A is also mixed with reaction solution B and buffer to form a reaction system of a specified volume; Placing the reaction system, standard solution, and reference substance on a PCR amplification instrument for amplification; The detection of target bacteria in the sample is confirmed based on the color development of the specific probe.
9. The method according to claim 8, characterized in that The sample is from at least one of feces, tissue, blood and body fluid; the tissue includes at least one of tumor tissue, non-tumor pathological tissue and healthy tissue.
10. The method according to claim 8, characterized in that The total DNA concentration of the sample mixed with each reaction solution A was 1×10 3 ~1×10 7 copies / mL range.
11. The method according to claim 8, characterized in that Each reaction system containing the total DNA of the sample is amplified simultaneously, and the amplification conditions are consistent.
12. The method according to claim 8, characterized in that The standard solution and the control substance present the specified results after amplification, which confirms that the current round of amplification is valid.