KR20220026367A - Composition for detection and identification of microorganisms associated with periodontal diseases - Google Patents

Abstract

The present invention relates to a composition for detecting bacteria causing oral diseases and a method for extracting nucleic acids using the same. To achieve the purpose of the present invention, the present invention provides a composition for simultaneous detection of oral disease-causing bacteria, which comprises: a first set of primers consisting of the oligonucleotides of SEQ ID NO: 11 and SEQ ID NO: 12; a second primer set consisting of the oligonucleotides of SEQ ID NO: 13 and SEQ ID NO: 14; a third primer set consisting of the oligonucleotides of SEQ ID NO: 15 and SEQ ID NO: 16; and a fourth primer set consisting of the oligonucleotides of SEQ ID NO: 17 and SEQ ID NO: 18, which specifically bind to nucleic acids obtained from biological samples taken from the oral cavity.

Description

Composition for detecting oral disease-causing bacteria

The present invention relates to a composition for detecting bacteria causing oral disease and a method for extracting nucleic acids using the same.

PCR (polymerase chain reaction) test is a technology that amplifies DNA by repeatedly duplicating a specific part of DNA. It is widely used in the diagnosis of diseases such as viral infections. Recently, using real-time PCR, it has reached a stage where quantitative analysis is possible. By directly examining the gene using PCR, which is a gene amplification technology, DNA or RNA containing the genetic information of a pathogen is extracted from the saliva or blood of a person infected with a virus or bacteria, and then amplified to confirm whether a disease is infected as a specific biomarker. It is molecular diagnostic technology that enables prevention and effective treatment through early diagnosis of diseases.

Among these molecular diagnostic technologies, the diagnostic technology using saliva has non-invasive, convenient, and efficient advantages compared to the conventional blood analysis method. A number of studies on its feasibility have been reported.

On the other hand, the oral cavity refers to a body organ that is connected from the lips to the pharynx start of the throat. The oral cavity is an entrance to the digestive tract and is a very important organ responsible for the function of ingesting food, the function of speech making sound, and the function of taste for feeling the taste of food. The oral cavity has good conditions for bacteria, fungi, viruses, and protozoa to inhabit. The oral cavity maintains proper temperature and humidity, and the presence of gingival sulcus fluid, epithelial cell fragments, saliva components, and food residues facilitates bacterial growth in the oral cavity. These bacteria living in the oral cavity are collectively called oral bacteria. Examples of representative oral bacteria include Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola. , Prevotella intermedia, Fusobacterium nucleatum, Parvimonas micra, Campylobacter rectus, Prevotella nigrescen nigrescens), Eubacterium nodatum, Eikenella corrodens, Streptococcus mitis, Streptococcus mutans, Streptococcus sobrinus sobrinus), Lactobacillus casei, Staphylococcus aureus, Enterococcus faecalis, Actinomyces viscosus, and the like.

The present invention reduces the time of detection and amplification of a target material in molecular diagnostic technology through saliva sample collection, so that it is possible to detect a target material within an examination time during dental treatment and to determine the presence of related bacteria and microorganisms. Provided are a composition for detecting oral disease-causing bacteria, which is the core of a dedicated field-type periodontal disease diagnosis system, and a nucleic acid extraction method using the same.

In order to achieve the object of the present invention, the present invention provides a first primer set comprising the oligonucleotides of SEQ ID NO: 11 and SEQ ID NO: 12, which specifically binds to a nucleic acid obtained from a biological sample collected from the oral cavity, SEQ ID NO: 13 and a second primer set consisting of the oligonucleotides of SEQ ID NO: 14, a third primer set consisting of the oligonucleotides of SEQ ID NO: 15 and SEQ ID NO: 16, and a fourth primer set consisting of the oligonucleotides of SEQ ID NO: 17 and SEQ ID NO: 18 It provides a composition for simultaneous detection of bacteria causing oral diseases.

In order to achieve the object of the present invention, the present invention provides a probe consisting of an oligonucleotide of SEQ ID NO: 19, a probe consisting of an oligonucleotide of SEQ ID NO: 20, and a probe consisting of an oligonucleotide of SEQ ID NO: 21. Bacteria causing oral diseases A composition for simultaneous detection of

In order to achieve another object of the present invention, the present invention provides an oligonucleotide for species-specific differentiation of periodontal disease-related bacteria comprising any one of the nucleotide sequences of SEQ ID NOs: 43 to 78 or a nucleotide sequence complementary thereto.

In order to achieve another object of the present invention, the primer set is Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Tre To provide a composition for simultaneous detection of oral disease-causing bacteria, specifically detecting any one or more oral disease-causing bacteria selected from the group consisting of Treponema denticola.

In order to achieve another object of the present invention, the first primer set is in the A gene region, the second primer set is in the B gene region, the third primer set is in the C gene region, and the fourth primer set is in the D gene region. To provide a composition for simultaneous detection of bacteria causing oral disease, which binds specifically.

According to the present invention, it is possible to provide a primer specifically designed for each causative organism for the detection of periodontal disease causative bacteria in a sample collected from the oral cavity.

In addition, according to the present invention, there is an effect that is very excellent in detection sensitivity and specificity compared to the existing PCR (Polymerase Chain Reaction) technique.

1 shows the experimental results of Aa Primer/probe set.
Figure 2 shows the probe concentration condition setting for each mix of Pg.
3 shows a sensitivity test for a positive sample.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

In addition, the embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art. Accordingly, the shapes and sizes of elements in the drawings may be exaggerated for clearer description, and elements indicated by the same reference numerals in the drawings are the same elements.

According to one embodiment of the present invention Aggregatibacter actinomycetem comitans (Aggregatibacter

actinomycetemcomitans), Porphyromonas gingivalis or Campylobacter rectus, which can complementarily bind to genomic genes, such as SEQ ID NOs: 1 to 30, detecting periodontal disease causative bacteria having the nucleotide sequence A primer is provided.

In order to enable simultaneous qualitative and quantitative analysis of the causative bacteria of periodontal disease, primer base pairs and probes capable of specifically binding to each of the causative bacteria of periodontal disease were designed as follows.

Aggregatibacter actinomycetemcomitans

Aa forward primer: 5'-ACA CGT GCT ACA ATG GCG TA-3' (SEQ ID NO: 1)

Aa reverse primer: 5'-TCA TAC CGT GGT AAA CGC CC-3' (SEQ ID NO: 2)

Aa probe: 5'-ACC AAC CAG CGA TGG GGA GTG AA-3' (SEQ ID NO: 3)

Porphyromonas gingivalis

Pg forward primer: 5'-GTG AAA ACC GTC TTC CCT TCG-3' (SEQ ID NO: 4)

Pg reverse primer: 5'-GGA TGT AAG GGC CGT GTT GAA-3' (SEQ ID NO: 5)

Pg probe: 5'-TGT AGG TGC TGC ATG GTT GTC GT-3' (SEQ ID NO: 6)

Tannerella forsythia

Tf forward primer: 5'-AAG CTC TAC AGC GAA AGC GT-3' (SEQ ID NO: 7)

Tf reverse primer: 5'-ACA ACC ATG CAG CAC CTA CA-3' (SEQ ID NO: 8)

Tf probe: 5'-CGG CAA CGG TGA AAC TCA AAG GA-3' (SEQ ID NO: 9)

Treponema denticola

Td forward primer: 5'-TGT TGG GTT AAG TCC CGC AA-3' (SEQ ID NO: 10)

Td reverse primer: 5'-TTG CGT TTT GCT TGA CCT CG-3' (SEQ ID NO: 11)

Td probe: 5'-TGG CGG AAC TGC CGA TGA CAA AT-3' (SEQ ID NO: 12)

According to one embodiment of the present invention, the genomic gene may be a 16S ribosomal RNA gene.

That is, among the bacteria causing periodontal disease, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, or Campylobacter lectus

(Campylobacter rectus), etc., using the 16S ribosomal RNA gene as a template, PCR can be performed by using the primer base pair capable of complementary binding to the gene.

Example 1

1.1 Primer Preparation

Selection of target strains

The four representative target strains that cause oral diseases are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, or Campylobacter by searching the prior literature. rectus) was selected.

1. 2 Primer Design

From each prior literature, the target region for each strain and the published primer sequence for each strain were confirmed. Thereafter, the name of each strain was searched in NCBI or BLAST to select the target nucleotide disclosure sequence (specific region within 1 kb), and blasting the published primer sequence was performed to compare the entire sequence with no similarity. sequence was selected.

For the selected sequence, the primer sequence was designed by selecting the same conditions for the primer temperature (58 degrees), probe temperature (63 degrees), and PCR product size (around 100 to 200 bp) using the primer 3 program.

For the designed primer sequence, specific detection for each target strain was verified by NCBI BLAST for the entire selected sequence.

By setting the actual experimental conditions, non-specific detection results for other strains other than the target bacteria of the primer set for each strain were cross-checked. Two sets of final primer sequences (set 1 and set 2) were confirmed.

Example 2

2.1 Working test of primer sets

PCR was performed using each of the primers of Example 2 for the target strain to confirm whether the primers prepared for each target strain work and whether the primers for a plurality of strains operate under the same conditions for simultaneous diagnosis. PCR master mix (2x AmpMaster Taq) used in the PCR reaction was purchased from GeneAll.

It was confirmed that the primer sets prepared by performing PCR using primer sets 1 and 2 prepared for each strain on the four strains worked properly for each strain under the same PCR conditions.

2.2 Cross-reaction test PCR composition of primer sets

PCR was performed by preparing a composition to test whether a primer specifically prepared for the target strain works (cross-reaction occurs) against other strains

After mixing the bacterial standard DNAs for the 4 strains, each primer was tested using 18 single primer set 1, and it was confirmed that each primer specifically detected only the corresponding strain in the 18 strain DNA mixture. . However, as a result of testing each of the primers using primer set 2 with respect to the strain DNA mixture, it was confirmed that the cross-reaction of the primers other than the target strain occurred in the strain mixture.

In addition, as a result of testing using single strain DNA after mixing each primer set 1 for the 4 strains, each primer set in the primer mixture works specifically for the target strain with respect to the standard DNA of 4 bacteria. was confirmed. However, after mixing each primer set 2 for the 4 strains, PCR was performed on the standard DNA of the 4 types of bacteria. Confirmed.

The above results show that accurate detection results cannot be confirmed only with prediction by NCBI BLAST for the primer sequence prepared using the primer production program, and primer set 1 for the four strains of the present invention is a primer mixture. It was confirmed that only each corresponding strain can be accurately detected even using

Example 3

3.1 Probe test

Real-time-PCR was performed to test the probes for detecting the four strains of the present invention. NEXproqTMPCR 2X MASTER MIX required for real-time PCR was purchased from Geneslabs (korea).

As a result of performing real-time-PCR on four strains, it was confirmed that the probe for each strain according to the present invention worked properly.

3.2 Multiplex Real-Time-PCR

Primers for 4 strains were mixed for each set (5 sets) to test whether they were detectable at the same time.

After mixing the DNA of the four strains and the primers for each strain for each set, multiplex real-time-PCR was performed. It was confirmed that the primer set mixture worked normally for the strain DNA mixture.

Example 4

sensitivity test

The sensitivity of the primer set according to the present invention was tested in real-time PCR against four strain plasmid DNA.

After preparing by diluting the concentration of the strain plasmid DNA at various magnifications, real-time-PCR was performed.

The sensitivity of all primer sets prepared against all four strains was high, and in particular, the sensitivity of the primer sets against the Ss strain was the highest.

Claims (4)

that specifically binds to a nucleic acid obtained from a biological sample taken from the oral cavity;
A first primer set composed of oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, a second primer set composed of oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4, and a third primer set composed of oligonucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 A composition for simultaneous detection of bacteria causing oral diseases, comprising a set, and a fourth primer set consisting of oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8. A composition for simultaneous detection of bacteria causing oral diseases, comprising a probe composed of the oligonucleotide of SEQ ID NO: 9, a probe composed of the oligonucleotide of SEQ ID NO: 10, and a probe composed of the oligonucleotide of SEQ ID NO: 11. The primer set consists of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. A composition for simultaneous detection of oral disease-causing bacteria, which specifically detects any one or more oral disease-causing bacteria selected from the group. a bacterium that specifically binds to the A gene region, the second primer set to the B gene region, the third primer set to the C gene region, and the fourth primer set to the D gene region. composition for simultaneous detection of

Images