KR100965289B1 - PCR method for identification of Bordetella pertussis from closely related Bordetella species using 16S rDNA and PCR primer therefor - Google Patents

Abstract

The present invention provides a PCR method, and PCR primers therefor for the differential diagnosis of VOR to VOR to telra-to-Peer Systems (Bordetella pertussis) from telra relatives. According to the present invention, a 16S-F2 / R1 (SEQ ID NO: 2 and SEQ ID NO: 3) primer set designed using the 16S rDNA gene was provided, and the pertussis causative organism from Bordetella myoma including B. holmesii . In Bordetella pertussis can be quickly and accurately differentiated.

Pertussis, PCR, Primer, Bordetella Pertussis, Bordetella Holmesi

Description

PCR method for identification of Bordetella pertussis from closely related Bordetella species using 16S rDNA and PCR primer therefor}

The present invention relates to a PCR technique for the differentiation of Bordetella pertussis from the Bordetella related species and to a PCR primer therefor. More specifically, the present invention provides a PCR capable of rapidly and accurately discriminating Bordetella pertussis from Bordetella species, including Bordetella holmesii , using 16S rDNA gene. Techniques and PCR primers therefor.

Pertussis is an acute disease caused by infection of the throat by B. pertussis, the causative bacterium, and is mainly an infectious disease in infants and young children. After infection, on average, seven days of incubation period, followed by the recovery of the three stages of clinical symptoms, catarrhal, mild, and recovery. Cataracts last for 1-2 weeks after onset and show mild cold symptoms such as runny nose, conjunctivitis, tears, mild cough and mild fever.Gyeonghaegi begins at the end of 2 weeks after onset and lasts about 2-4 weeks. It lasts for more than 1 minute and is accompanied by vomiting and mucus sputum at the end of the cough. Coughing respiratory distress may also cause cyanosis. The recovery phase is the period when the coughing and recovery frequency gradually decreases over 1-2 weeks. Pertussis deaths are known to be mainly due to respiratory complications, in particular pneumonia is a major cause of about 54% of pertussis deaths. Other major complications include acute encephalopathy, otitis media, otitis malnutrition and dehydration, pneumothorax caused by paroxysmal cough, hernia, nasal bleeding, and subdural hemorrhage (Chapter 8 Pertussis in VPD Surveillance) Manual (3rd Edition), 2002, CDC).

As mentioned above, whooping cough is a respiratory disease caused by bacterial infection, especially in infants. The high incidence of pertussis was reported until the 1980s (1,928,384 cases / year), but the incidence was gradually reduced by the vaccination movement that began in the 1940s. However, local outbreaks of pertussis have been reported between 1990 and 2000 in well-vaccinated countries such as the United States, the Netherlands, Japan and Australia (de Melker HE, Schellekens JFP, Neppelenbroek SE, Mooi FR, Rumke HC, Conyn-van). Spaendonck MAE: Reemergence of pertussis in the highly vaccinated population of the netherlands: Observations on surveillance data.Emer Inf Dis, 2000, 6: 348-357 ; de Melker HE, Conyn-van Spaendonck MAE, Rumke HC, van Wijngaarden JK, Mooi FR, Schellekens JFP: Pertussis in the netherlands: an outbreak despite high levels of immunization with whole-cell vaccine. Emer Inf Dis, 1997, 3: 175-178; Mooi FR, van Loo IHM, King AJ: Adaptation of Bordetella pertussis to vaccination: A cause for its reemergence? Emer Inf Dis, 2001, 7: 526-528. Although much effort has been made to determine the cause of this increase, it is not yet clear. Therefore, the diagnostic process will play an important role in preventing the local onset of whooping cough at an early stage.

Laboratory diagnosis of whooping cough is currently carried out with effective methods of culture and PCR (Chapter 8 Pertussis in VPD Surveillance Manual (3rd Edition), 2002, CDC; WHO, Laboratory manual for the diagnosis of whooping cough caused by Bordetella pertussis / Bordetella parapertussis , WHO / IVB / 04.14, 2004). Usually, a clear standard of diagnosis is to identify pathogens by culture. In general, however, pertussis bacteria grow significantly slower than other pathogenic microorganisms, and the size of the colonies formed is small. In addition, PCR testing is very important in pertussis diagnosis service because causative organisms may be killed during transportation of specimens. In particular, the PCR diagnostic test can confirm positive and negative within a few hours, so the results can be confirmed in a short time compared to the culture test is important for the initial determination.

To date, various diagnostic primer sets have been developed for diagnosing whooping cough. Among these primer sets, BP1 / 2 for factor IS481 (Fry NK, Tzivra O, Li YT, McNiff A, Doshi N, Maple PA, Crowcroft NS, Miller E, George RC, Harrison TG: Laboratory diagnosis of pertussis infections: the role of PCR and serology.J Med Microbiol 2004, 53: 519-525; van der Zee A, Agterberg C, Peeters M, Schellekens J, Mooi FR: Polymerase chain reaction assay for pertussis: simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis . J Clin Microbiol 1993, 31: 2134-2140), PTp1 / p2 for the promoter region of Pertussis toxin (Fry NK, Tzivra O, Li YT, McNiff A, Doshi N, Maple PA, Crowcroft NS, Miller E, George RC, Harrison TG: Laboratory diagnosis of pertussis infections: the role of PCR and serology.J Med Microbiol 2004, 53: 519-525; Birkebaek NH, Heron I, Skjodt K: Bordetella pertussis diagnosed by polymerase chain reaction. APMIS 1994, 102: 291-294; Houard S, Hackel C, Herzog A, Bollen A: Specific identification of Bordetella pertussis by the polymerase chain reaction. Res Microbiol 1989, 140: 477-487), P1 / 2/3 at the top of the Porin gene (Li Z, Jansen DL, Finn ™, Halperin SA, Kasina A, O'Connor SP, Aoyama T, Manclark CR, Brennan MJ: Identification of Bordetella pertussis infection by shared-primer PCR. J Clin Microbiol 1994, 32: 783-789). In particular, due to its high sensitivity and specificity, the BP primer set utilizing the IS481 sequence is the most widely used pertussis diagnostic primer (WHO, Laboratory manual for the diagnosis of whooping cough caused by Bordetella pertussis / Bordetella parapertussis , WHO / IVB / 04.14, 2004).

However, despite the high sensitivity of BP primers, Bordetella holmesii ) has recently been reported. More specifically, the IS481 element has recently been found to exist not only in B. pertussis but also in B. holmesii (Loeffelholz MJ, Thompson). CJ, Long KS, Gilchrist MJ: Detection of Bordetella holmesii using Bordetella pertussis IS481 PCR assay. J Clin Microbiol 2000, 38: 467; Poddar SK: Detection and discrimination of B. pertussis and B. holmesii by real-time PCR targeting IS481 using a beacon probe and probe-target melting analysis. Mol Cell Probes 2003, 17: 91-98; Reischl U, Lehn N, Sanden GN, Loeffelholz, MJ: Real-Time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii . J Clin Microbiol 2001, 39: 1963-1966; Templeton KE, Scheltinga SA, van der Zee A, Diederen BMW, Kruijssen AM, Goossens H, Kuijper E, Claas ECJ: Evaluation of real-time PCR for detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for clinical diagnosis. J Clin Microbiol 2003, 41: 4121-4126).

Bordetella Holmesii was discovered in 1995 and has been known to be unrelated to respiratory disease until 1998, but was found in 33 patients with pertussis-like symptoms in Massachusetts in 1995-1998. Associated with the disease (Yih WK, Silva EA, Ida J, Harrington N, Lett SM, George H: Bordetella holmesii -like organisms isolated from Massachusetts patients with pertussis-like symptoms. Emerg Infect Dis 1999, 5: 441-443; Mazengia E, Silva EA, Peppe JA, Timperi R, George H: Recovery of Bordetella holmesii from patients with pertussis-like symptoms: use of pulsed-field gel electrophoresis to characterize circulating strains. Bordetella hall in addition 2330-2333), and Bordetella-to-Peer Systems (B. pertussis), Bordetella para-to-Peer Systems (B. parapertussis) as one of the causes of atypical pneumonia microorganisms: J Clin Microbiol 2000, 38 Messi has to include a (B. holmesii) (Acta Pharmacologica Sinica , 24 (12): 1308-1313, 2003), according to the information presented at the annual meeting of the ASM 2005 (annual meeting), in Virginia (Virginia) The occurrence of disease caused by Bordetella Holmesii was investigated and announced, along with Bordetella pertussis among 9 species of Bordetella, Bordetella Bronquicept Utica (B. bronchiseptica), Bordetella para-to-Peer systems (B. parapertussis), Bordetella hole mesh (B. holmesii) has stated that is associated with respiratory tract infections in humans and other mammals and Bordetella hole mesh (B. holmesii) recently bronchial sense of the human being found in And it has been reported to be associated with the paper (CLINICAL MICROBIOLOGY REVIEWS, 18 (2): 326-382, 2005). Thus Bordetella hole mesh (B. holmesii) is believed to be closely related to the human respiratory disease.

Bordetella Holmesii is a species of the genus Bordetella that showed the highest similarity with B. pertussis in 16S rDNA sequencing (Yih WK, Silva EA, Ida J). , Harrington N, Lett SM, George H: Bordetella holmesii -like organisms isolated from Massachusetts patients with pertussis-like symptoms.Emerg Infect Dis 1999, 5: 441-443). Specific primers of Bordetella holmesii for diagnosis and differentiation have not been reported yet, and BP primers are currently being applied for detection of B. holmesii (WHO, Laboratory manual) for the diagnosis of whooping cough caused by Bordetella pertussis / Bordetella parapertussis , WHO / IVB / 04.14, 2004).

Therefore, under the PCR diagnostic system using the BP primer set for pertussis testing, it is necessary to confirm the false positive of Bordetella holmesii for the specimens that are positive for the BP primer. Diagnosis of pertussis-like disease caused by detella holmesh infection is also needed. For this reason, there is a Bordetella-to-Peer Systems (B. pertussis) and Bordetella hole mesh (B. holmesii) need to be able to differential diagnosis primer to diagnostic methods developed in the diagnostic PCR in step.

The present invention has been made to solve the problems of the prior art, Bordetella pertussis from the Bordetella cultivars including Bordetella holmesii using 16S rDNA gene The present invention relates to a PCR technique capable of quickly and accurately discriminating a) and a PCR primer for the same.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, the present invention provides a PCR primer set for detecting Bordetella pertussis consisting of 16S-F2 primer (SEQ ID NO: 2) and 16S-R1 primer (SEQ ID NO: 3). to provide.

The present invention provides a PCR primer set for Bordetella holmesii ( B. holmesii ) detection consisting of 16S-F1 primer (SEQ ID NO: 1) and 16S-R1 primer (SEQ ID NO: 3).

Also provides a PCR method for detecting the present invention is Bordetella Peer-to-cis (B. pertussis) or Bordetella hole mesh (B. holmesii) comprising the steps of PCR amplification using the primer set do.

Hereinafter, with reference to the accompanying drawings will be described in more detail with respect to the present invention.

The inventors of the present invention are Bordetella bronchiseptica ( B. bronchiseptica ), Bordetella hinge ( B. hinzii ), Bordetella Holmesii ( B. holmesii ) , Bordetella parapertussis ( B. parapertussis), Bor Five 16S rDNA sequences of B. pertussis were downloaded from NCBI GenBank and sorted by a multiple alignment method, and the parts showing differences in the sequences were searched.

As a result, as shown in FIG. 1, the strains of the 661th and 782th nucleotide sequences were identified. Based on this, PCR primers were designed as follows, and Bordetella Holmesii ( B. holmesii) was designed. The present invention was completed by constructing a primer combination capable of distinguishing primers specific to B. pertussis and three other Bordetella spp.) And Bordetella pertussis . Table 1 shows the PCR primers and specific combinations designed by the present invention.

Designed PCR  Primers ( Designed PCR primers ) 16S-F1 5'-AACTGCATTTTTAACTACCGA-3 '(SEQ ID NO: 1) 16S-F2 5'-AACTGCATTTTTAACTACCGG-3 '(SEQ ID NO: 2) 16S-R1 5'-ATCCTGTTTGCTCCCCACA-3 '(SEQ ID NO: 3) 16S-R2 5'-ATCCTGTTTGCTCCCCACG-3 '(SEQ ID NO: 4) PCR  Specificity ( PCR specificity ) B. holmesii 16S-F1 x 16S-R1 B. pertussis 16S-F2 x 16S-R1 B. bronchiseptica , B. parapertussi s,
B. hinzii
16S-F2 x 16S-R2

16S-F1 / 16S- designed for the purpose of detecting Bordetella holmesii ( B, holmesii) as a result of confirming whether the primer sets produced as described above according to the present invention can be discriminated as desired. The R1 primer set did not respond to other species. Also Bordetella chevron kisep urticae (B. bronchiseptica) and Bordetella para-to-Peer system detecting the 16S-F2 / 16S-R2 primer set designed for the (B. parapertussis) is Bordetella Peer-to-cis (B. pertussis ) Did not show reactivity. In addition, had only indicate a positive response Bordetella Peer-to-cis (B. pertussis) a primer set of 16S-F2 / 16S-R1 is only Bordetella Peer-to-cis (B. pertussis) is designed to detect, for VOR There was no response to B. bronchiseptica and B. parapertussis (see FIG. 3).

16S-F2 / R1 primer set that can be used for diagnosing whooping cough designed by the present invention can be identified up to 5 pg / reaction using purified chromosomal DNA as a PCR template, and 10 ⁵ cells / for cell suspension. Detection up to the concentration of ml was possible (see FIGS. 4 and 5). In addition, the 16S-F2 / R1 primer set was tested in the positive and negative clinical specimens only positive results in clinical specimens of pertussis positive patients confirmed that it can be applied to the clinical specimens (see Figure 6).

Furthermore, the PCR primer sets according to the present invention may be used in a PCR technique in one or more combinations and included in a kit so that the causative agents of respiratory diseases may be detected quickly and accurately at the same time.

As described above, the present invention is to provide a diagnostic Vor primer set for the five species of the genus is important to telra telra Bor-to-Peer system Bor to telra hole mesh (B. pertussis), designed based on the 16S rDNA sequence (B . holmesii), Bordetella para-to-Peer systems (B. parapertussis), Bordetella chevron kisep urticae (B. bronchiseptica), Bordetella hinge (B. hinzii) to reduce the effects that can be diagnosed by PCR can do.

In particular, the 16S primer set (16S-F2 / R1) according to the present invention has no cross-reactivity unlike the conventional BP primer set, so that Bordetella Holmesii ( B. holmesii) and Bordetella pertussis ( B. pertussis ) can be distinguished quickly and accurately.

Furthermore, according to the present invention, a primer set (16S-F1 / R1) specific to Bordetella holmesii, which has been mainly used for biochemical tests and cellular fatty acid analysis, has been provided. The advantage of quickly identifying Detella Holmesii is that have.

Therefore, the present invention will be referred to as a useful invention that can quickly and accurately detect and diagnose the main causative organisms from the respiratory specimens of the similar respiratory disease caused by Bordetella genus.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Materials and methods ( Materials and Methods )

1. Strain

The reference strains used in the present invention are Bordetella Peer-to-cis (Bordetella pertussis; ATCC9797), Bordetella hole mesh (Bordetella holmesii ; ATCC 51541, Bordetella parapertussis ; ATCC15237), Bordetella bronchiseptica ; ATCC 31124). In addition to these reference strains, nasopharyngeal inhalation clinical specimens collected from whooping cough patients were used for the confirmation test.

2. PCR  Preparation of the template

4 Bordetella Genomic DNA was purified from the cultured cells of the species and used as a PCR template. To this end, all reference strains were incubated for 5-7 days at 37 ° C. without cephalexin in Regan-Lowe medium. After incubation, strain cells were scraped and genomic DNA was isolated using commercial kits as directed by the manufacturer (QIAmp DNA Mini Kitm QIAGEN).

For clinical specimens, collected nasopharyngeal aspirates were used as templates for direct PCR reactions. A portion of the sample (0.5-1 ml) was heated in boiling water for 5 minutes. After centrifugation at 15,000 rpm for 5 minutes, 1-2 μl of supernatant was used as a PCR template.

3. PCR primer

Four PCR primers were designed from 16S rDNA sequences of five Bordetella species ( B. holmesii , B. bronchiseptica, B. pertussis , B. parapertussis , B. hinzi ). DNA sequence information of 16S rDNA gene was downloaded from GenBank of NCBI. The nucleotide sequences thus collected were aligned using multiple sorting by the MEGA program, and regions containing different nucleotides were analyzed using the “Sequence Output” program and PCR primers were designed (FIG. 1). For reference, the BP primer sequence (BP1: 5'-GAT TCA ATA GGT TGT ATG CAT GGT T-3 '(SEQ ID NO: 5), BP2: 5'-TTC AGG CAC ACA AAC TTG ATG GGC G-3' (SEQ ID NO: 5) No. 6) is cited from published papers (Reischl U et al. J Clin Microbial 39: 1963-6, 2001).

4. PCR  Condition

Standard reaction mixture (20 μl) contains 2 μl of 10-fold buffer, 1 μl of dNTPs mixture (2.5 mM each), 1 μl of F-primer (10 pmol / μl), 1 μl of R-primer (10 pmol) , 0.25 μl of SP-Taq polymerase (2.5 Unit / μl), 1 μl of template (purified genomic DNA or bacterial suspension), and distilled water were added to make a total of 20 μl of reaction solution. For 16S primer sets, 5 ng of purified genomic DNA was used and dimethylsulfoxide (DMSO) was added to the reaction mixture to a final concentration of 2.5%.

Temperature conditions for the 16S primer set were 10 times denatured at 35 times 98 ° C, 15 at 64 ° C (16S-F1 / R1) or 66 ° C (16S-F2 / R1 and 16S-F2 / R2) depending on the primer set. Two-step PCR conditions were applied with the binding and stretching steps for a second. For the BP primer set, 35 cycles of 95 ° C. 5 seconds denaturation and 54 ° C. 10 seconds binding and stretching steps were performed.

Example  One. BP primer  Set of B. holmesii Cross-reaction with

The cross-reactivity of BP primers against B. holmesii and other 2 Bordetella species ( B. bronchiseptica and B. parapertussis ) was confirmed in the present invention. As shown in FIG. 2, the positive bands appeared simultaneously in two lanes (lanes 4 and 5) of Bordetella pertussis and B. holmesii . However, B. parapertussis (lane 2) and B. brochiseptica (lane 3) were not detected by the BP primer set. Therefore, the use of the BP primer set currently widely used in PCR diagnosis of pertussis should be considered false positive by Bordetella holmesii .

Example 2. PCR primer  Design of sets

In the present invention, the inventors considered the 16S rDNA gene to develop a new primer set for pertussis PCR diagnosis. To this end, five major Bordetella species ( B. pertussis , B. holmesii , B. bronchiseptica , B. parapertussis) and B. hinzi ) 16S rDNA sequences were collected from GenBank of NCBI, and sorted by multiple sorting method to identify sequence variations. As a result, there were species-specific differences in the two positions of the 661th and 782th nucleotide sequences in the aligned aggregates. We chose these two positions as motifs for PCR primer design, and based on this we designed four new primers. Primers designed as shown in Table 1 differ only in their 3'-terminal nucleotides.

Example 3. 16S engineered PCR primer  Specificity of the set

As mentioned above, the specificity of the designed new primer set was examined for four Bordetella species ( B. pertussis , B. holmesii , B. parapertussis , and B. bronchiseptica ) For specific amplification, these four primers Combinations were applied: 16S-F1 / 16S-R1 set for B. holmesii, 16S-F2 / 16S-R1 for B. pertussis , other Borde telra used the 16S-F2 / 16S-R2 set for (Bordetella) species (Table 1). after the reaction, the amplified target band was confirmed by a 1.5% agarose gel electrophoresis. As a result, a combination of primer set Bordetella Peer-to-cis (B. pertussis), Bordetella hole mesh (B. holmesii), Bordetella para-to-Peer systems (B. parapertussis), Bordetella chevron kisep urticae (B. bronchiseptica) using Confirm that the specific amplification of Was., A positive PCR band is only shown only in each of the target species (lane 2, lane 5, lane 11 and lane 12). Therefore, PCR primers are each Bordetella (Bordetella) designed species as shown in Fig. 3 Specificity was shown.

In particular, 16S-F2 and 16S-R1 primers were applied for Bordetella pertussis . As shown in Figure 3, the positive band is only Bordetella Peer-to-cis (B. pertussis), was detected only in samples (lane 5). However, it was also confirmed that nonspecific bands were also detected in samples of Bordetella holmesii under relaxed PCR conditions. Therefore, the use of 16S-F2 and 16S-R1 primers requires stringent PCR conditions. For this reason, DMSO was added to the reaction mixture to a final concentration of 2.5%.

Example  4. Designed 16S PCR primer  Sensitivity of the set

The sensitivity of the 16S-F2 / R1 primer set to Bordetella pertussis was evaluated using purified genomic DNA and cell suspension. Under fixed PCR conditions, detection limits of the target bands were first examined using purified genomic DNA. After the reaction, 10 reactants were electrophoresed on 1.5% agarose gel and the amplified bands were observed under UV illuminator. As a result, as shown in Figure 4, when using genomic DNA, the detection limit was 5 pg / reaction (lane 2 of Figure 4).

In the case of cell suspension, 10 were heated for ^4-10 suspension ¹⁰ cells / mL of the cells in boiling water for 5 minutes, it was centrifuged at 15,000 rpm for 5 minutes. After centrifugation, 1 μl supernatant was added to the PCR reaction mixture as a template. After 1.5% agarose gel electrophoresis, the amplified PCR bands were observed by UV irradiator. As a result, as shown in Fig. 5, the detection limit of the 16S rDNA gene according to the present invention was> 10 ⁵ cells / ml in the cell suspension (lane 6 in Fig. 5).

Example  5. Application to clinical specimens

The applicability of the 16S primers according to the invention on clinical specimens was evaluated. The samples were nasopharyngeal aspirates collected from suspected whooping cough in 2006. These samples have already been tested positive for B. pertussis by BP primer PCR and culture. These samples were heat treated under the same conditions as the cell suspension experiment of Example 4, PCR with a 16S-F2 / R1 primer set, and then observed with a UV irradiator by 1.5% agarose gel electrophoresis, as shown in FIG. 6, All positive samples were also confirmed positive by the 16S-F2 / R1 PCR primer set (Figure 6, lanes 1-4). Therefore, the newly developed 16S-F2 / R1 PCR primer set according to the present invention will be applicable to actual clinical specimens confirming the infection of pertussis.

1 is a Bordetella hole mesh (B. holmesii), Bordetella chevron kisep urticae (B. bronchiseptica), Bordetella para-to-Peer Systems (B. parapertussi s), Bordetella Peer-to-cis (B. pertussis ) And multiple alignment of 16S rDNA sequence of Bordetella hinge ( B. hinzii ).

Figure 2 is a Bordetella hole mesh (B. holmesii) of BP primer set according to the prior art and Bordetella Peer-to-cis a diagram showing the cross-reactivity of the (B. pertussis).

3 shows specific amplification of each Bordetella spp. Using 16S primer sets (16S-F1 / R1, 16S-F2 / R1, 16S-F2 / R2) according to the present invention. .

Figure 4 is a view of the check of the detection sensitivity of the Bordetella-to-Peer Systems (B. pertussis) by the 16S primer set (16S-F2 / R1) according to the invention using the purified genomic DNA results.

5 is a Bordetella Peer-to-cis a diagram showing a result of comparing the detection sensitivity of (B. pertussis) by the 16S primer set (16S-F2 / R1) according to the present invention using a cell suspension liquid.

Figure 6 is a view showing the results of applying the PCR primer set (16S-F2 / R1) according to the invention to a clinical sample.

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Claims (8)

16S-F2 primer (SEQ ID NO: 2) and 16S-R1 primer (SEQ ID NO: 3) Bordetella Peer-to-cis (B. pertussis), the detection primer set for PCR consisting of a. 16S-F1 primer (SEQ ID NO: 1) and 16S-R1 primer (SEQ ID NO: 3) Bordetella hole mesh (B. holmesii) detecting PCR primer sets consisting of. 16S-F2 primer (SEQ ID NO: 2) and 16S-R2 primer (SEQ ID NO: 4) Bordetella chevron kisep urticae (consisting of Bordetella bronchiseptica ), Bordetella parapertussi s) or Bordetella hinge (Bordetella hinzii ) PCR primer set for detection. A PCR method for detecting Bordetella pertussis comprising the step of amplifying by PCR using the primer set according to claim 1. The method of claim 4, wherein dimethylsulfoxide (DMSO) is added to the reaction mixture so that the final concentration is 2.5%. A PCR method for detecting Bordetella holmesii , comprising amplifying by PCR using the primer set according to claim 2. PCR amplification using the primer set of claim 3, characterized in that it comprises Bordetella bronchiseptica ( B. bronchiseptica ), Bordetella parapertussis ( B. parapertussi s) or Bordetella PCR method for detecting the hinge ( B. hinzii ). A kit for detecting and diagnosing Bordetella species, a respiratory bacterium comprising at least one PCR primer set selected from the group consisting of the following primer sets; (a) a primer set for PCR consisting of 16S-F2 primer (SEQ ID NO: 2) and 16S-R1 primer (SEQ ID NO: 3); (b) a primer set for PCR consisting of 16S-F1 primer (SEQ ID NO: 1) and 16S-R1 primer (SEQ ID NO: 3); And (c) A primer set for PCR consisting of 16S-F2 primer (SEQ ID NO: 2) and 16S-R2 primer (SEQ ID NO: 4).

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