JP4806749B2 - Bordetella pertussis gene detection method using LAMP method and primer set used in this method - Google Patents

Description

  The present invention relates to a pertussis gene detection method using the LAMP method and a primer set used in this method.

  Pertussis is an acute respiratory infection in children caused by infection with Bordetella pertussis. The number of whooping cough patients worldwide is estimated at 20-40 million per year, and the number of deaths is estimated at about 200-400,000. The affected individuals are mainly pre-vaccinated and unvaccinated infants. Based on the number of reported cases, the estimated annual number of cases in Japan is 28,000 in 2000 and 15,000 in 2001. Bacteriological or serological diagnosis is necessary for the definitive diagnosis of pertussis, but diagnosis (clinical diagnosis) is mainly performed in clinical practice based on clinical symptoms. The reason for this is that since the definitive diagnosis methods by bacterial isolation and antibody titer measurement require several days or more of examination days, post-diagnosis is performed at the clinical site. Clinical diagnosis is easy for patients with typical whooping cough symptoms such as long-term cough, but there is a problem that it cannot be diagnosed for patients with early symptoms of infection with atypical symptoms.

In recent years, genetic diagnosis has been widely introduced as a test method for infectious diseases, and PCR targeting the insertion sequence (IS481) of the pertussis and the pertussis toxin gene (ptx) has been studied as a laboratory diagnostic method even in pertussis. . (Houard et al., Res Microbiol, 140: 477-487, 1989 (Non-Patent Document 1), Dragsted et al., J Med Microbiol, 53: 749-754, 2004 (Non-Patent Document 2))
Houard et al., Res Microbiol, 140: 477-487, 1989 Dragsted et al., J Med Microbiol, 53: 749-754, 2004

  However, although the laboratory diagnostic method using PCR has a high positive rate compared with the bacterial culture test, it is still not sufficient, and the development of a more sensitive and simple diagnostic technique has been desired. In recent years, the LAMP method (Loop mediated isothermal Amplification) that enables simple and rapid diagnosis has been developed as a genetic diagnosis method that replaces the PCR method, and its application research is being promoted in many infectious diseases. The LAMP method does not require a gene amplification device essential for PCR reaction, has an advantage that it can be determined in a short time, and further enables visual determination by adding a fluorescent dye to the reaction system. Therefore, it has been attracting attention as an inspection technology that can be introduced into laboratories such as hospitals where equipment is not sufficiently developed. However, since a LAMP primer for Bordetella pertussis has not been developed, it was necessary to design and evaluate a LAMP primer that specifically detects Bordetella pertussis.

  Therefore, an object of the present invention is to provide a primer sequence that can detect a Bordetella pertussis gene with high sensitivity and specificity by the LAMP method, and to provide a method for detecting a Bordetella pertussis gene using this primer sequence.

  Furthermore, an object of the present invention is to provide a Bordetella pertussis gene detection kit using the above primer sequence.

The present invention for solving the above problems is as follows.
[1] A primer set capable of amplifying at least a part of the Bordetella pertussis gene by the LAMP method is prepared, DNA is extracted from the specimen, and the extracted DNA is subjected to an amplification operation by the LAMP method using the primer set. A method for detecting a Bordetella pertussis gene contained in a sample, comprising confirming whether the amplified DNA is contained in the product later ,
The method, wherein four primers comprising the base sequences of SEQ ID NOs: 1 to 4 shown in the sequence table and two loop primers comprising the base sequences of SEQ ID NOs: 5 to 6 shown in the sequence table are used as the primer set .
[ 2 ] The method according to [ 1 ], wherein whether the amplified product contains amplified DNA is confirmed by measuring the turbidity of the amplified product or using a fluorescent dye.
[ 3 ] Four primers consisting of the base sequences of SEQ ID NOs: 1 to 4 shown in the sequence table, which are used for detecting the Bordetella pertussis gene contained in the bacterial cells in the specimen by the LAMP method, are shown in the sequence table. A primer set comprising two loop primers consisting of the nucleotide sequences of SEQ ID NOs: 5-6.
[ 4 ] A kit for detecting a Bordetella pertussis gene contained in a sample by the LAMP method, comprising the primer set according to [ 3 ].

  According to the present invention, a Bordetella pertussis gene can be detected with high sensitivity and specificity. The LAMP method is a technique that can be introduced into a laboratory such as a hospital because it does not require complicated operations like the PCR method. Therefore, according to the present invention, simple and quick pertussis diagnosis is possible in the clinical field. In recent years, it has been pointed out that adults may be a source of infection for infants by carrying Bordetella pertussis. When an adult carries Bordetella pertussis, clinical diagnosis is extremely difficult because it does not show symptoms similar to infants. Therefore, the present invention enables pertussis diagnosis not only for infants but also for adults.

The present invention relates to a method for detecting a Bordetella pertussis gene contained in bacterial cells in a specimen. This method includes the following steps.
(1) Prepare a primer set that can amplify at least part of the Bordetella pertussis gene by the LAMP method.
(2) DNA is extracted from the specimen.
(3) The extracted DNA is subjected to an amplification operation by the LAMP method using the primer set.
(4) After the amplification operation, it is confirmed whether or not the amplified DNA is contained in the product.

[Primer set]
In the method of the present invention, a primer set capable of amplifying at least a part of the Bordetella pertussis gene by the LAMP method is prepared. Bordetella pertussis is a bacterium belonging to the genus Bordetella. Similar bacteria belonging to the genus Bordetella include parapertussis and bronchial septic bacteria. Therefore, the present inventors searched for a specific sequence for the Bordetella pertussis gene with these bacteria. As a result, as shown in FIG. 1, the pertussis toxin promoter sequence of Bordetella pertussis found that there was a different sequence from other Bordetella genus bacteria (parapertussis, bronchial septic bacteria). A LAMP primer was designed for the same sequence. The LAMP primer design support software (PrimerExplorer Ver.3, Eiken Chemical Co., Ltd.) was used for the design, and the present inventors considered seven possible candidates from the designed primer set (162 types). A primer set was selected. Then, the selected primer sets were synthesized, and the detection sensitivity and specificity of each primer set were examined using DNA purified from various Bordetella bacteria. As a result, it was found that the primer set of the present invention (ID2 in the examples) showed the highest sensitivity, and did not detect Bordetella bacteria (5 strains, 8 strains) other than Bordetella pertussis (very high specificity). did.

  An object of the present invention is to reliably detect only the Bordetella pertussis gene despite the presence of many types of Bordetella bacteria similar to Bordetella pertussis. From the viewpoint of achieving such an object, as a primer set capable of amplifying at least part of the Bordetella pertussis gene by the LAMP method, a primer set consisting of the following four types of DNAs of SEQ ID NOs: 1 to 4 should be used. Is preferred.

  The base sequence (part) of the Bordetella pertussis gene is shown in FIG. The base sequence shown in FIG. FIG. 2 shows the relationship between the base sequence of the Bordetella pertussis gene and each DNA of the primer set of the present invention. By using a primer set consisting of four types of DNAs of SEQ ID NOs: 1 to 4, a partial DNA of the Bordetella pertussis gene can be amplified by the LAMP method. Although the minimum unit of DNA to be amplified is calculated to be 156 bp, in the LAMP method, DNA is amplified like a cauliflower, so that genes of various sizes other than 156 bp are actually amplified.

BP-F3 (F3): 5'-CCGCATACGTGTTGGCA-3 '(SEQ ID NO: 1)
BP-B3 (B3c): 5'-TGCGTTTTGATGGTGCCT-3 '(SEQ ID NO: 2)
BP-FIP (F2-F1c):
5'-TTGGATTGCAGTAGCGGGATGTGCATGCGTGCAGATTCGTC-3 '(SEQ ID NO: 3)
BP-BIP (B1-B2c):
5'-CGCAAAGTCGCGCGATGGTAACGGATCACACCATGGCA-3 '(SEQ ID NO: 4)

  In the method of the present invention, each of the four types of primers has the entire sequence shown above, and when the pertussis gene is present in the sample, the pertussis gene is reliably amplified regardless of the strain type. It is preferable from the viewpoint. However, even if each primer has a part of its bases deleted or substituted, or has other bases added, there may be those that function as a similar primer. That is, it has a base sequence having a deletion, substitution and / or addition of 1 to several bases in any one of SEQ ID NOs: 1 to 4, and a part of the Bordetella pertussis gene is amplified by the LAMP method. A primer set containing a DNA having a primer function that can be used as part or all of the DNA of the primer set can also be used in the method of the present invention.

The primer set of the present invention may further contain the following DNA (loop primer) in addition to the four types of DNA.
BP-LF (LFc): 5'-ACGGAAGAATCGAGGGTTTTGTAC-3 '(SEQ ID NO: 5)
BP-LB (LB): 5'-GTCACCGTCCGGACCGTG-3 '(SEQ ID NO: 6)

  In the method of the present invention, even if the primer set does not include the loop primer, at least a part of the Bordetella pertussis gene can be amplified by the LAMP method. However, it is preferable to include the loop primer because the amplification time can be greatly shortened.

  Even in the above-mentioned loop primer, there may be one that exhibits a function as a loop primer, even if one of the bases of each primer is deleted or substituted, or another base is added. That is, when the nucleotide sequence of SEQ ID NO: 5 or 6 has a nucleotide sequence having a deletion, substitution and / or addition of 1 to several nucleotides, and at least a part of the Bordetella pertussis gene is amplified by the LAMP method A primer set including a DNA having a loop primer function as a part of the primer set can also be used in the method of the present invention.

  The range of “1 to several” in the “base sequence having deletion, substitution and / or addition of 1 to several bases” as used herein is not particularly limited. , Preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, particularly preferably 1.

  Each DNA constituting the primer set of the present invention can be appropriately obtained by a known DNA preparation method based on the base sequence.

  Although the present invention relates to a method for detecting a Bordetella pertussis gene using the above primer set, the present invention includes the above primer set itself as an invention. Furthermore, the present invention also includes a kit for detecting the Bordetella pertussis gene contained in the bacterial cells in the specimen, which contains the above-described primer set of the present invention, by the LAMP method.

  The primer set of the present invention can be, for example, in a form containing the four or six types of primers (DNA) in an amount necessary for a single amplification of a gene by the LAMP method. The amount of primer (DNA) necessary for the gene amplification by one LAMP method is, for example, in the range of 5 to 40 pmol / reaction tube.

[DNA extraction]
In the method of the present invention, DNA is extracted from a specimen. Specifically, DNA is directly extracted from a strain in a specimen using a commercially available DNA extraction kit. Examples of the DNA extraction kit include QIAamp DNA Micro Kit (Cat. No. 56301, Qiagen).

[Amplification by LAMP method]
In the method of the present invention, the extracted DNA (DNA template) is subjected to an amplification operation by the LAMP method using the primer set. The amplification operation by the LAMP method can be performed as follows, for example. A reagent (for example, Bst DNA polymerase, reaction buffer, dNTPs, primer set, distilled water) is mixed, dispensed into a reaction tube, and then a DNA template (extracted DNA) is added. Next, for example, incubation is performed at 65 ° C. The incubation time is, for example, in the range of 30 minutes to 90 minutes.

[Confirmation of amplified DNA]
In the method of the present invention, it is confirmed whether the amplified DNA is contained in the product after the amplification operation. When the pertussis gene is contained in the bacterial cells contained in the specimen, the pertussis gene is amplified by the amplification operation. On the contrary, if the pertussis gene is not contained in the microbial cells contained in the specimen, there is no DNA amplified by the amplification operation.

  Whether or not the amplified product contains amplified DNA can be confirmed, for example, by measuring the turbidity of the amplified product or by visual fluorescence detection. Specifically, the turbidity of the amplification product can be measured as follows. The turbidity of the final amplification product is measured over time with a real-time turbidity measuring device or with a turbidity measuring device. The fluorescent visual detection can be carried out by adding a fluorescent visual reagent for reaction, and visually observing the fluorescent color of the reaction solution by applying a UV lamp.

  The LAMP detection system using the primer set of the present invention showed the maximum sensitivity at a reaction temperature of 65 ° C., and its detection limit was 10 fg / tube for Bordetella pertussis DNA. This amount of DNA corresponds to 2.3 copies of Bordetella pertussis genomic DNA. When comparing the detection sensitivity with the existing PCR method (PTp1 / p2-PCR, IS481-PCR), the detection limit of IS481-PCR, which is considered to be the highest sensitivity, is 1,000 fg / tube, and the primer set ID2 of the present invention The LAMP detection system using (SEQ ID NOs: 1 to 6) was found to be about 100 times more sensitive than IS481-PCR. IS481-PCR has the disadvantage of detecting other Bordetella bacteria, while PTp1 / p2-PCR has the disadvantage of poor specificity (> 100 pg / tube) although it has excellent specificity.

The present invention includes the primer set described in the above method. Furthermore, the present invention also includes a kit including the primer set of the present invention. What is contained in a kit can be the following things, for example.
1.Bst DNA polymerase
2. Reaction buffer
3.dNTPs
4.Primer set (primer of the present invention)
5. Control DNA (positive control)
6. Reaction tube
7. Manual

  As described above, in the method of the present invention, the presence or absence of DNA by-products can be determined by turbidity measurement or visual fluorescence detection. In the case of detecting with fluorescent visual observation, a fluorescent visual inspection reagent can be included in the kit. When using a turbidity measuring device, the fluorescent visual detection reagent is not included in the kit.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Strain
For confirmation of LAMP primer specificity, Bordetella pertussis, B. parapertussis, B. bronchiseptica R05, B. hinzii ATCC51730, B. holmesii ATCC51541, B. avium ATCC35086 Six bacterial species (a total of 17 strains) were tested. Bordetella pertussis has 9 strains of B. pertussis Higashihama strain, Yamaguchi strain and National Institute of Infectious Diseases (BP256-260, BP289-290). 4 strains of BPP01) were used.

DNA preparation
Bordetella bacteria were cultured on Bordet-Gengou agar medium at 36 ° C. for 1-2 days, and then DNA was extracted and purified from the cells. Commercially available Qiagen genomic tip 20 / G (Qiagen) was used for purification, and the concentration of the obtained DNA was determined from the absorbance at a wavelength of 260 nm.
In this example, purified DNA is required to evaluate the specificity and sensitivity of the LAMP primer, so the above shows the method for preparing the DNA used for the evaluation. However, in actual tests (for example, clinical specimens), DNA can be extracted directly from the specimen without culturing the bacteria.

PCR
The sensitivity and specificity of the LAMP primer set was carried out by comparing with existing PCR methods (PTp1 / p2-PCR, IS481-PCR). PTp1 / p2-PCR follows the method of Houard et al (Houard et al., Res Microbiol, 140: 477-487, 1989), and IS481-PCR is Dragsted et al (Dragsted et al., J Med Microbiol, 53: 749-754). , 2004). The presence or absence of gene amplification was confirmed by ethidium bromide staining after separating PCR products by 3% agarose gel electrophoresis.

Design of LAMP primer Since a pertussis toxin promoter sequence of Bordetella pertussis has a sequence different from other Bordetella bacteria, a LAMP primer was designed for the same sequence (FIG. 1). For design, LAMP primer design support software (Primer Explorer Ver.3, Eiken Chemical) was used, and 7 candidate primer sets (ID1 to 7) were selected from the designed primer sets. Since primer set ID2 showed the highest sensitivity in preliminary experiments, specificity evaluation was performed only for primer set ID2. Primer synthesis was requested from Hokkaido System Science Co., Ltd., and HPLC purification grade primers were used.

  FIG. 1 shows a comparison of Bordetella bacteria pertussis toxin promoter sequences. The pertussis toxin promoter sequences of B. pertussis (SEQ ID NO: 7), B. bronchiseptica (SEQ ID NO: 8), and B. parapertussis (SEQ ID NO: 9) were compared. At base numbers -186 to -177 in the sequence shown in FIG. 1, deletion of the gene is observed in Bordetella pertussis and Bacterial sepsis. Furthermore, the presence of different bases is confirmed between the pertussis and bronchial septic bacteria around the sequence. From this sequence comparison, a LAMP primer was designed for the same region. The coding region indicates the translation region of pertussis toxin S1 subunit.

LAMP detection system
The detection sensitivity and specificity of the LAMP primer set were evaluated using Loopamp DNA amplification reagent kit (Eiken Chemical). The LAMP detection system was prepared in 25 μl of reaction solution with 0.2 μM of each outer primer (BP-F3, BP-B3), 1.6 μM of each inner primer (BP-FIP, BP-BIP), 0.8 μM of each loop primer ( BP-LF, BP-LB), 1 μl of Bst-DNA polymerase, 12.5 μl of 2 × reaction solution, and 1 μl of DNA solution were reacted at 65 ° C. for 1 hour. Then, the reaction stop process for 2 minutes was performed at 80 degreeC. A Loopamp reaction tube (Eiken Chemical) was used as the reaction tube, and the increase in turbidity associated with gene amplification was measured using a real-time measurement device (Loomamp LA-320C, Eiken Chemical). The amplification of the gene was also confirmed by 2% agarose gel electrophoresis. Table 1 shows the reaction composition of the LAMP detection system.

  FIG. 3 shows the results of the sensitivity of the LAMP detection system using primer set ID2. Purified Bordetella pertussis DNA was serially diluted and the detection limit of the LAMP detection system was measured. A) is an increase curve of turbidity caused by gene amplification reaction. The increase in turbidity was measured using a real-time measuring device (Loopamp LA-320C, Eiken Chemical). B) shows the result of analyzing the amplification product after 60 minutes of reaction by agarose gel electrophoresis.

Table 2 shows a comparison of sensitivity between the LAMP method using the primer set ID2 and the PCR method. Sensitivity comparison with the existing PCR method (PTp1 / p2-PCR, IS481-PCR) was performed using purified Bordetella pertussis DNA. The detection limits of LAMP method, PTp1 / p2-PCR, and IS481-PCR were 10, 1 × 10 ⁵ , and 1,000 fg / tube, respectively.

Table 3 shows the results of the specificity test of the LAMP detection system for Bordetella bacteria. Specificity against Bordetella pertussis and other Bordetella bacteria (5 species, 8 strains) was examined. LAMP and PTp1 / p2-PCR were shown to detect all Bordetella pertussis and not other Bordetella bacteria. On the other hand, IS481-PCR was shown to detect some parapertussis and B. holmesii. Although it has already been reported that B. holmesii has the IS481 gene, this clinical strain is the first to confirm the presence of Parapertussis with IS481.

  The LAMP detection system using the primer set according to the present invention can be expected to be put into practical use as an in vitro diagnostic agent for pertussis infection.

Comparison of pertussis toxin promoter sequences of Bordetella bacteria. The design position of primer set ID2 of the present invention relative to the pertussis toxin promoter sequence of Bordetella pertussis is shown. LFc and LB indicate loop primers, and the amplification region by LAMP method is from F2 to B2c. Sensitivity test result of LAMP detection system using primer set ID2.

Claims (4)

Prepare a primer set that can amplify at least part of the Bordetella pertussis gene by LAMP method, extract DNA from the sample, use the extracted DNA for amplification operation by LAMP method using the primer set, A method for detecting a Bordetella pertussis gene contained in a specimen comprising: confirming whether or not the amplified DNA is contained therein ,
The method, wherein four primers comprising the base sequences of SEQ ID NOs: 1 to 4 shown in the sequence table and two loop primers comprising the base sequences of SEQ ID NOs: 5 to 6 shown in the sequence table are used as the primer set . 2. The method according to claim 1, wherein whether or not the amplified product contains amplified DNA is confirmed by measuring the turbidity of the amplified product or using a fluorescent dye. Four primers consisting of the nucleotide sequences of SEQ ID NOs: 1 to 4 shown in the sequence table and SEQ ID NOs: shown in the sequence table, which are used for detecting the Bordetella pertussis gene contained in the bacterial cells in the specimen by the LAMP method A primer set comprising two loop primers consisting of 5 to 6 nucleotide sequences. A kit for detecting a Bordetella pertussis gene contained in a specimen by the LAMP method, comprising the primer set according to claim 3 .