CN114480688A

CN114480688A - Periodontal pathogen nucleic acid detection kit and application

Info

Publication number: CN114480688A
Application number: CN202210141059.7A
Authority: CN
Inventors: 赵俊
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-05-13

Abstract

The invention discloses a periodontal pathogen nucleic acid detection kit and application thereof, and the periodontal pathogen nucleic acid detection kit can be used for detecting actinobacillus actinomyces actinomycetemcomitans, porphyromonas gingivalis, Fostainer, prevotella intermedia and treponema denticola in a sample, and is beneficial to diagnosis and treatment of periodontitis. By adopting the nucleic acid detection kit for the periodontal pathogens, a plurality of bacteria related to periodontitis can be identified at one time, the workload is greatly reduced, and the detection efficiency is improved.

Description

Periodontal pathogen nucleic acid detection kit and application

Technical Field

The invention particularly relates to a periodontal pathogen nucleic acid detection kit and application.

Background

The oral microflora contains more than 700 microorganisms, most of the bacteria are normal oral flora, only a few bacteria are closely related to the occurrence and development of periodontal disease, one or more dominant bacteria can be isolated in lesion areas of various types of periodontal disease, have obvious virulence or pathogenicity, can interfere host defense capacity through various mechanisms, and have the potential of initiating periodontal destruction, and are called as periodontal pathogens.

Periodontal pathogens are the major causes of periodontal disease and are also important factors affecting oral health. Oral microbiota is a key factor in health or disease. Periodontal pathogens cause ecological disturbances of the luminal flora and can lead to oral and systemic diseases. Dysbiosis of the oral cavity is associated with systemic diseases such as cardiovascular diseases, cancer and diabetes.

Periodontitis is a chronic and multifactorial disease; it is related to dietary habit, smoking and drinking, genetic inheritance, oral health care and mental stress. Caused by a specific group of periodontal pathogenic bacteria; the imbalance of oral microbial flora leads to the long-term and massive breeding of pathogenic bacteria. The main characteristic is inflammation; usually painless, but if left untreated, can lead to progressive destruction of the periodontal tissue with subsequent tooth loss.

Bacteria are the main cause of periodontitis: the bacteria responsible for the development of periodontitis are primarily anaerobic and gram-negative bacteria. These interactions are close and occur at different stages of the disease.

Healthy periodontal tissue is primarily colonized by aerobic gram-positive "beneficial" bacteria. These form part of the normal local flora and help to maintain a stable oral condition. Anaerobic/facultative anaerobic, gram-negative periodontal disease pathogens are present even in individuals with healthy periodontal tissues, albeit in small numbers. These marker bacteria become a problem when their concentration rises helically due to poor oral hygiene or reduced immunocompetence.

The colonization of the dental socket by periodontal bacteria occurs in different stages, each with the appearance of a specific species and associated symptoms. After initial colonization by moderately pathogenic microorganisms, an aggressive inflammatory process develops, with clinical manifestations determined by highly pathogenic species. It is noteworthy that periodontitis is a chronic infectious disease, and bacteria can spread to people around patients.

Periodontitis is caused by various bacteria, which differ significantly in their properties and characteristics. The sensitivity of different species to particular antibiotics also varies. The periodontitis-associated bacteria are mainly accompanied by Actinobacillus Actinomycetemcomitans (AA), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fostainers (Tf), Treponema denticola (Td), and the like.

Currently, there is no kit for the detection of periodontal pathogens as described above in the prior art.

Disclosure of Invention

Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a nucleic acid detection kit for periodontal pathogens and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

a primer and a probe for detecting actinobacillus actinomycetemcomitans, porphyromonas gingivalis, Fostanemia furiosus, Prevotella intermedia and treponema denticola, wherein the primer and the probe comprise:

detecting actinobacillus actinomycetemcomitans DNA primer and probe:

a first upstream primer: 5'-GCTAGGTAACATCGGTAA-3'

First downstream primer: 5'-GCACGATCAAATTGTTTC-3'

A first probe: 5 '-FAM-CTGATTGCCAAGCTGACCACA-BHQ 1-3'

Detecting DNA primers and probes of Porphyromonas gingivalis:

a second upstream primer: 5'-ATGCCTAAGACCAATACTTA-3'

A second downstream primer: 5'-GCCTGAATGCTATAAGAAG-3'

A second probe: 5 '-VIC-ACGCTTCCTGCTTCTCTGCC-BHQ 1-3'

Detecting Fostainer DNA primers and probes:

a third upstream primer: 5'-CCTGTAGTAGAACGATATCTC-3'

A third downstream primer: 5'-CGAGAGTACGCTTAATCC-3'

A third probe: 5 '-ROX-TTCGTCCGTCTCCATCAACTGC-BHQ 2-3'

Detecting the DNA primers and the probes of the intermediate prevotella:

a fourth upstream primer: 5'-CACACGCTGGCGAAACCTAC-3'

A fourth downstream primer: 5'-CACGTGGCGTTGCTTCTTTC-3'

A fourth probe: 5 '-FAM-CCGAAGATGCGCCGTTGAAC-BHQ 1-3'

DNA primers and probes for detecting treponema denticola:

a fifth upstream primer: 5'-AGAAAGGCTTTGGGCGACAG-3'

A fifth downstream primer: 5'-GCTGGAGCCGTAGCTTCCAT-3'

A fifth probe: 5 '-VIC-CGGGTCCTCACCCGCTCTTC-BHQ 1-3'.

Further, the primers and the probes also comprise internal reference primers and probes,

internal reference primers and probes:

a sixth upstream primer: 5' -TGGAGCATGTGGTTTAATTCGA-3

A sixth downstream primer: 5'-ACGAGCTGACGACAACCATG-3'

A sixth probe: 5 '-Cy 5-TGG + TAAGG + TTCTTCGCGT-BHQ 2-3'

The base is preceded by a "+" indicating that the base is an LNA modification.

A nucleic acid detection kit for periodontal pathogens comprises the primers and the probes.

Further, the kit also comprises a PCR detection mixed solution.

Further, the PCR detection mixed solution comprises Tris-HCl, KCl and MgCl₂，NH₄Cl, CA-63, glycerol 5%, DMSO, dNTP, UDG enzyme and Taq DNA polymerase.

Further, the PCR assay mixture includes 20mM Tris-HCl, 50mM KCl, 3mM MgCl₂，5mM NH₄Cl, CA-630.05%, glycerol 5%, DMSO, 5%, dNTPs 500nM each, UDG enzyme 10U, Taq DNA polymerase 5U.

Further, after the primers and the probes are added into the PCR detection mixed solution, the concentration of each primer is 100-500 nM, and the concentration of each probe is 50-200 nM. The primer and the probe are the primer and the probe.

Further, the kit also comprises a positive control substance and/or a negative control substance. The positive reference substance is actinobacillus actinomycetemcomitans, porphyromonas gingivalis, Fostainarum, Prevotella intermedia and treponema denticola plasmid containing a target amplification sequence, and the negative reference substance is RNA enzyme-removed water.

The primers and the probes for detecting actinomycetemcomitans actinobacillus, porphyromonas gingivalis, Fostainer bacteria, prevotella intermedia and treponema denticola are applied to the preparation of the detection reagent for the periodontal pathogens.

The application of the periodontal pathogen nucleic acid detection kit in preparing a periodontal pathogen detection reagent.

The invention has the beneficial effects that:

(1) the nucleic acid detection kit for the periodontal pathogens can detect actinobacillus actinomycetemcomitans, porphyromonas gingivalis, forsythiasis, prevotella intermedia and treponema denticola in a sample, and is favorable for diagnosis and treatment of periodontitis.

(2) By adopting the nucleic acid detection kit for the periodontal pathogens, a plurality of bacteria related to periodontitis can be identified at one time, the workload is greatly reduced, and the detection efficiency is improved.

Drawings

FIG. 1 is a sensitivity amplification curve of Actinobacillus actinomycetemcomitans.

FIG. 2 is a graph of the sensitivity amplification of Porphyromonas gingivalis.

FIG. 3 is a graph showing the sensitivity amplification curve of Treponema denticola.

FIG. 4 is a graph showing the sensitivity amplification curve of Prevotella intermedia.

FIG. 5 is a sensitivity amplification curve of Fostainer.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments provided in the present application without any inventive step are within the scope of protection of the present application.

Reference in the specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the specification. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of ordinary skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments without conflict.

Unless defined otherwise, technical or scientific terms referred to herein shall have the ordinary meaning as understood by those of ordinary skill in the art to which this application belongs. Reference to "a," "an," "the," and similar words throughout this application are not to be construed as limiting in number, and may refer to the singular or the plural. The present application is directed to the use of the terms "including," "comprising," "having," and any variations thereof, which are intended to cover non-exclusive inclusions; reference to "connected," "coupled," and the like in this application is not intended to be limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. Reference herein to "a plurality" means greater than or equal to two. "and/or" describes an association relationship of associated objects, meaning that three relationships may exist, for example, "A and/or B" may mean: a exists alone, A and B exist simultaneously, and B exists alone. Reference herein to the terms "first," "second," "third," and the like, are merely to distinguish similar objects and do not denote a particular ordering for the objects.

The reagents and instruments used in the following examples are commercially available, where 1M is 1 mol/L.

A primer and a probe for detecting nucleic acid of periodontal pathogenic bacteria, wherein the primer and the probe comprise:

DNA primers and probes for detecting Actinobacillus actinomycetemcomitans:

a first upstream primer: 5'-GCTAGGTAACATCGGTAA-3' (SEQ ID No.1)

First downstream primer: 5'-GCACGATCAAATTGTTTC-3' (SEQ ID No.2)

A first probe: 5 '-FAM-CTGATTGCCAAGCTGACCACA-BHQ 1-3' (SEQ ID No.3)

DNA primers and probes for detecting Porphyromonas gingivalis:

a second upstream primer: 5'-ATGCCTAAGACCAATACTTA-3' (SEQ ID No.4)

A second downstream primer: 5'-GCCTGAATGCTATAAGAAG-3' (SEQ ID No.5)

A second probe: 5 '-VIC-ACGCTTCCTGCTTCTCTGCC-BHQ 1-3' (SEQ ID No.6)

The DNA primers and probes for detecting the Fostainer are as follows:

a third upstream primer: 5'-CCTGTAGTAGAACGATATCTC-3' (SEQ ID No.7)

A third downstream primer: 5'-CGAGAGTACGCTTAATCC-3' (SEQ ID No.8)

A third probe: 5 '-ROX-TTCGTCCGTCTCCATCAACTGC-BHQ 2-3' (SEQ ID No.9)

The DNA primers and probes for detecting the intermediate prevotella:

a fourth upstream primer: 5'-CACACGCTGGCGAAACCTAC-3' (SEQ ID No.10)

A fourth downstream primer: 5'-CACGTGGCGTTGCTTCTTTC-3' (SEQ ID No.11)

A fourth probe: 5 '-FAM-CCGAAGATGCGCCGTTGAAC-BHQ 1-3' (SEQ ID No.12)

DNA primers and probes for detecting Treponema denticola:

a fifth upstream primer: 5'-AGAAAGGCTTTGGGCGACAG-3' (SEQ ID No.13)

A fifth downstream primer: 5'-GCTGGAGCCGTAGCTTCCAT-3' (SEQ ID No.14)

A fifth probe: 5 '-VIC-CGGGTCCTCACCCGCTCTTC-BHQ 1-3' (SEQ ID No.15)

In some preferred modes, the primers and the probes further comprise internal reference primers and probes,

internal reference primers and probes:

a sixth upstream primer: 5' -TGGAGCATGTGGTTTAATTCGA-3 (SEQ ID No.16)

A sixth downstream primer: 5'-ACGAGCTGACGACAACCATG-3' (SEQ ID No.17)

A sixth probe: the base of 5 '-Cy 5-TGG + TAAGG + TTCTTCGCGT-BHQ 2-3' (SEQ ID No.18) is preceded by "+" indicating that the base is an LNA modification. Here, the Tm value of the probe was increased by LNA modification.

In some preferred modes, the kit for detecting the nucleic acid of the periodontal pathogen comprises the primer and the probe.

In some preferred modes, the kit further comprises a PCR detection mixture.

In some preferred embodiments, the PCR assay mixture comprises Tris-HCl, KCl, MgCl₂，NH₄Cl, CA-63, glycerol 5%, DMSO, dNTP, UDG enzyme and Taq DNA polymerase.

In some preferred embodiments, the PCR assay mix comprises 20mM Tris-HCl, 50mM KCl, 3mM MgCl₂，5mM NH₄Cl, CA-630.05%, glycerol 5%, DMSO 5%, dNTPs 500nM each, UDG enzyme 10U, Taq DNA polymerase 5U.

In some preferred embodiments, the concentration of each primer is 100-500 nM and the concentration of each probe is 50-200 nM after the primers and probes are added to the PCR assay mixture. The primer and the probe are the primer and the probe.

In some preferred modes, the kit further comprises a positive control and/or a negative control. In some embodiments, the positive control is an actinobacillus actinomycetemcomitans, porphyromonas gingivalis, forsteria, prevotella intermedia, and treponema denticola plasmid comprising the amplification sequence of interest, which are commercially available or can be prepared. In other embodiments, the positive control is a solution containing actinobacillus actinomycetemcomitans, porphyromonas gingivalis, mestrania, treponema denticola, and prevotella intermedia positive nucleic acids. Of course, in other embodiments, other forms of positive controls may be employed. The negative control is RNA enzyme-removed water.

The amplification and detection system comprises the following components:

1) PCR reaction System 1

Quadruple fluorescent quantitative PCR amplification and detection of actinobacillus actinomycetemcomitans, porphyromonas gingivalis and Fostainarum.

A 30 microliter PCR reaction system comprising: 20mM Tris, 50mM KCl, 3mM MgCl₂，5mM NH₄Cl, CA-630.05% (V/V), glycerol 5% (V/V), DMSO 5% (V/V), dNTPs 500nM each, primers 200nM each, probes 100nM each, UDG enzyme 10U, Taq DNA polymerase 5U.

The primer comprises: detecting actinobacillus actinomycetemcomitans DNA primers, detecting porphyromonas gingivalis DNA primers and probes, and detecting fosfomycin DNA primers and internal reference primers;

the probes comprise a probe for detecting actinobacillus actinomycetemcomitans, a probe for detecting porphyromonas gingivalis, a probe for detecting fosfomycin and an internal reference probe.

2) PCR reaction System 2

Triple fluorescence quantitative PCR amplification and detection of Neprieria intermedius and Treponema denticola.

A 30 microliter PCR reaction system comprising: 20mM Tris, 50mM KCl, 3mM MgCl₂，5mM NH₄Cl, CA-630.05% (V/V), glycerol 5% (V/V), DMSO 5% (V/V), dNTPs 500nM each, primers 200nM each, probes 100nM each, UDG enzyme 10U, Taq DNA polymerase 5U;

the primer comprises: detecting a DNA primer of the prevotella intermedia, and detecting a DNA primer of the treponema denticola and an internal reference primer; the probes comprise a probe for detecting the prevotella intermedia, a probe for detecting the treponema denticola and a probe for detecting the internal reference.

3) PCR amplification procedure:

2 minutes at 50 ℃, so that aerosol pollution is prevented; heat-activated DNA polymerase at 95 ℃ for 2 minutes; 15 seconds at 95 ℃ and 40 seconds at 55 ℃ (fluorescence collected); for a total of 40 cycles.

The PCR amplification program is adopted in both the PCR reaction systems 1 and 2.

4) And (3) judging the effectiveness of the kit:

negative control: FAM/VIC/ROX has no amplification curve, and Cy5 has an amplification curve Ct < 30; the agent is effective;

positive control: FAM/VIC/ROX has an amplification curve, and Ct is less than 25;

the two requirements are met, the reagent is effective, and the next analysis can be carried out.

5) And (4) analyzing results:

FAM, VIC and ROX channels, Ct <35, the result is positive, and corresponding values are output;

FAM, VIC and ROX channels were not detected, and the result was negative.

(II) detection sensitivity by using the kit of the invention

The different kinds of periodontal pathogen positive plasmids include actinobacillus actinomycetemcomitans plasmid, porphyromonas gingivalis plasmid, treponema denticola plasmid, prevotella intermedia plasmid and fosetalin plasmid; adding RNase Free Water into the plasmid dry powder for dissolving to obtain a plasmid solution, and then diluting according to a 10-fold concentration gradient to obtain the concentration of the plasmid solution: 10⁸copies/ml，10⁷copies/ml，10⁶copies/ml，10⁵copies/ml，10⁴copies/ml，10³copies/ml，10²copies/ml。

Extracting nucleic acid from different kinds of periodontal disease positive plasmids with different concentrations, adding 20 μ L of nucleic acid extract into 30 μ L of PCR reaction system 1 and PCR reaction system 2, covering tube cap, mixing, centrifuging, loading, and amplifying by the above amplification procedure. The results of the sensitivity measurements are shown in FIGS. 1-5.

FIG. 1 is a sensitivity amplification curve of Actinobacillus actinomycetemcomitans; from left to right in the graph, the corresponding actinobacillus actinomycetemcomitans plasmid solution concentrations are, in order: 10⁸copies～10²copies/mL。

FIG. 2 is a graph of the sensitivity amplification of Porphyromonas gingivalis; from left to right in the graph, the corresponding concentration of the plasmid solution of Porphyromonas gingivalis is 10⁸copies～10²copies/mL。

FIG. 3 is a graph of sensitivity amplification of Treponema denticola; from left to right in the graph, the concentration of the plasmid solution of Treponema denticola is 10⁸copies～10²copies/mL。

FIG. 4 is a graph of the sensitivity amplification of Prevotella intermedia; from left to right in the graph, the corresponding concentration of the plasmid solution of Prevotella intermedia is 10⁸copies～10²copies/mL。

FIG. 5 is a sensitivity amplification curve for Fostainer; from left to right, the concentration of the plasmid solution of Fostaurinia is 10⁸copies～10²copies/mL。

From fig. 1 to 5 it can be derived: the kit has high sensitivity of detecting actinobacillus actinomycetemcomitans, porphyromonas gingivalis, Fostanemia furiosa, Prevotella intermedia and treponema denticola, and the sensitivity is 10²copies/ml。

(III) specific embodiment

Collecting 4mL of saliva samples of patients in a certain oral hospital according to the specification by adopting a saliva collector; the method is used for nucleic acid detection.

1. Sample treatment:

1) sample treatment 1:

1mL of saliva sample was centrifuged at 13000rpm for 5 minutes, and the supernatant was removed. Then, 1ml of physiological saline was added thereto to wash the mixture once, and the supernatant was removed after centrifugation at 13000rpm for 5 minutes. Adding 200 microliter of physiological saline, and extracting nucleic acid by a nucleic acid extraction instrument after resuspension.

Here the saliva sample, after being processed by the sample processing step, can be used for the detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Statina fosfomi.

2) Sample treatment 2:

a200. mu.l saliva sample was taken and the nucleic acid was extracted directly with a nucleic acid extraction reagent. Here the saliva sample, after being treated with a sample treatment step, can be used for the detection of Prevotella intermedia and Treponema denticola.

2. Loading and detecting:

1) preparation of reagents: taking out the reagent in the nucleic acid detection kit and dissolving at normal temperature (freezing and dissolving);

2) preparing a reagent: preparing a fluorescent PCR reaction system according to the sample number + 2; preparing a reaction system according to the number of the tubes by using the PCR reaction systems 1 and 2;

3) sample adding: respectively taking 20 microliters of nucleic acid samples, respectively adding the nucleic acid samples into 30 microliters of PCR reaction systems 1 and 2, tightly covering a tube cover, mixing uniformly, then performing instantaneous centrifugation, and loading on a machine.

4) The amplification procedure was set up under the following reaction conditions.

2 minutes at 50 ℃, and aerosol pollution is prevented; DNA polymerase heat activation at 95 ℃ for 2 minutes; 95 ℃, 15 seconds, 55 ℃, 40 seconds (fluorescence collected); for a total of 40 cycles.

3. And (4) analyzing results:

TABLE 1 sample test results

As can be seen from the data in Table 1, the detection results of the sample 1 are positive for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia, the detection results of the sample 2 are positive for Porphyromonas gingivalis, Prevotella intermedia and Treponema tartarum, the detection results of the sample 3 are positive for Porphyromonas gingivalis, Prevotella intermedia and Treponema tartarum, the detection results of the sample 4 are positive for Actinobacillus actinomycetemcomitans and the detection results of the sample 5 are positive for Treponema tartarum and Treponema furiosum. The above described test results are consistent with known clinical test results and coincide with the periodontal inflammation pattern.

The features of the above-described embodiments may be combined in any combination, and for the sake of brevity, all possible combinations of the above-described embodiments will not be described, but rather, the scope of the present disclosure should be considered as limited only by the combinations of the features described herein.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of protection. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A primer and a probe for detecting actinobacillus actinomycetemcomitans, porphyromonas gingivalis, Fostanemia furiosus, Prevotella intermedia and treponema denticola are characterized in that the primer and the probe comprise:

detecting actinobacillus actinomycetemcomitans DNA primer and probe:

a first upstream primer: 5'-GCTAGGTAACATCGGTAA-3'

First downstream primer: 5'-GCACGATCAAATTGTTTC-3'

A first probe: 5 '-FAM-CTGATTGCCAAGCTGACCACA-BHQ 1-3'

Detecting DNA primers and probes of Porphyromonas gingivalis:

a second upstream primer: 5'-ATGCCTAAGACCAATACTTA-3'

A second downstream primer: 5'-GCCTGAATGCTATAAGAAG-3'

A second probe: 5 '-VIC-ACGCTTCCTGCTTCTCTGCC-BHQ 1-3'

Detecting Fostainer DNA primers and probes:

a third upstream primer: 5'-CCTGTAGTAGAACGATATCTC-3'

A third downstream primer: 5'-CGAGAGTACGCTTAATCC-3'

A third probe: 5 '-ROX-TTCGTCCGTCTCCATCAACTGC-BHQ 2-3'

Detecting the DNA primers and the probes of the intermediate prevotella:

a fourth upstream primer: 5'-CACACGCTGGCGAAACCTAC-3'

A fourth downstream primer: 5'-CACGTGGCGTTGCTTCTTTC-3'

A fourth probe: 5 '-FAM-CCGAAGATGCGCCGTTGAAC-BHQ 1-3'

Detecting Treponema denticola DNA primers and probes:

a fifth upstream primer: 5'-AGAAAGGCTTTGGGCGACAG-3'

A fifth downstream primer: 5'-GCTGGAGCCGTAGCTTCCAT-3'

A fifth probe: 5 '-VIC-CGGGTCCTCACCCGCTCTTC-BHQ 1-3'.

2. The primer and probe for detecting actinobacillus actinomycetemcomitans, porphyromonas gingivalis, Fostanemia fortunei, Prevotella intermedia and Treponema denticola of claim 1, wherein the primer and probe further comprise an internal reference primer and probe,

internal reference primers and probes:

a sixth upstream primer: 5' -TGGAGCATGTGGTTTAATTCGA-3

A sixth downstream primer: 5'-ACGAGCTGACGACAACCATG-3'

A sixth probe: 5 '-Cy 5-TGG + TAAGG + TTCTTCGCGT-BHQ 2-3'

The base is preceded by a "+" indicating that the base is an LNA modification.

3. A kit for detecting nucleic acid of periodontal pathogen, comprising the primer and probe according to any one of claims 1 to 2.

4. The kit for detecting nucleic acid of periodontal pathogen according to claim 3, further comprising a PCR detection mixture.

5. The periodontal pathogen nucleic acid detection kit according to claim 4, wherein the PCR detection mixture comprises Tris-HCl, KCl, MgCl₂，NH₄Cl, CA-63, glycerol, DMSO, dNTP, UDG enzyme and Taq DNA polymerase.

6. The periodontal pathogen nucleic acid detection kit according to claim 3, wherein the kit further comprises a positive control and/or a negative control.

7. Use of the primers and probes for detecting actinobacillus actinomycetemcomitans, porphyromonas gingivalis, fosfomycin, prevotella intermedia and treponema denticola according to any one of claims 1 to 2 in the preparation of a reagent for detecting periodontal pathogens.

8. Use of the nucleic acid detection kit for periodontal pathogenic bacteria according to any of claims 3 to 6 for the preparation of a reagent for detecting periodontal pathogenic bacteria.