JP3914914B2 - Probe for diagnosis of infections caused by Streptococcus pyogenes - Google Patents

Description

The present invention relates to a probe useful for detecting and identifying a causative bacterium of an infectious disease, particularly Streptococcus pyogenes , which is a causative bacterium such as pharyngitis, rheumatic fever, nephritis, erysipelas, scarlet fever and sepsis.

  Diseases caused by infection with pathogenic microorganisms are collectively referred to as infectious diseases. Pathologically, infection refers to pathogenic microorganisms (hereinafter referred to as “fungi”) entering the body and establishing a foothold for growth. In other words, the onset due to the growth of bacteria in a living body depends on the interrelationship between the resistance of the host and the virulence of the bacteria.

  Streptococcus is a facultative or obligate anaerobic Gram-positive cocci that are arranged in a chain. Depending on the nature of the hemolytic ring surrounding the colony grown on the blood agar medium, it is divided into three types, α, β, and γ, and further, from A to V depending on the antigenicity of the C polysaccharide (Lancefield classification) It is classified into 20 groups (except I and J).

Streptococcus pyogenes ( Streptococcus pyogenes ) is a type of streptococci belonging to group A according to the Lancefield classification, showing β-type hemolysis (complete hemolysis), human pharyngitis, tonsillitis, scarlet fever, erysipelas, puerperium It is clinically important as a causative bacterium such as fever and sepsis. It is also known that after infection, secondary diseases such as rheumatic fever and nephritis, which are allergic diseases called poststreptococcal diseases, are known. Furthermore, in recent years, cases (fulminant group A streptococcal infection) accompanied by myositis and severe septic shock due to infection with Streptococcus pyogenes have been reported.

  The pharyngitis caused by Streptococcus pyogenes is often accompanied by marked pharyngeal redness and cervical lymphadenopathy, and the frequency of complaining of sore throat is high, so it is possible to suspect this disease from clinical symptoms. However, it may not always show clear clinical symptoms, and it is necessary to use optimal chemotherapy to prevent subsequent complications. At the same time, it is desirable to avoid unnecessary antibiotics. Rapid bacteriological diagnosis is required.

  In addition, fulminant group A streptococcal infection has been reported to be associated with severe necrotizing fasciitis in more than 50% of cases, and rapidly progressed to multi-organ failure, which may take a turning point in death. Not a few.

  In general, Streptococcus pyogenes is known to be highly sensitive to β-lactams such as ampicillin and cefaclor, but erythromycin has about 30% highly resistant strains and ofloxacin resistant strains appear. Because there are reports that it is, it is necessary to be careful when using macrolides and new quinolones.

  As described above, it is important to conduct an accurate early diagnosis and select an optimal antibacterial agent in an infectious disease derived from Streptococcus pyogenes.

  In normal biological methods, (1) examination of clinical symptoms, (2) culture of specimens, and (3) isolation and identification of Streptococcus pyogenes from the culture are essential. The treatment policy is decided only after it is done.

  For the identification of Streptococcus pyogenes, specimens are usually smeared and cultured on blood agar plates supplemented with 5% defibrinated blood such as sheep and horses, and the properties of the hemolysis ring around the colonies grown on the medium are observed. To do.

  However, in practice, it is usually difficult to determine the causative bacteria. That is, the shape of the colony varies greatly depending on the culture conditions, and identification is often difficult. Moreover, in addition to having a long time for culturing bacteria, further 3 to 4 days of culturing is required before obtaining the results of drug sensitivity results, and rapid diagnosis is impossible. In addition, if a large amount of antibiotics is administered at the time when infection is suspected, even if bacteria are contained in the sample, the increase or proliferation may be suppressed. The possibility of culturing bacteria from these specimens is extremely low.

  Furthermore, as a method as a subroutine, an instrumental analysis method of bacterial cell components and bacterial metabolites (Non-patent Document 1), a method using a specific antibody (Patent Document 1), and a high-level using the specificity of DNA There are methods by hybridization (Patent Document 2) and the like, but in any case, isolation of bacteria and culture of enrichment are essential.

On the other hand, focusing on the function of phagocytic cells in infectious diseases, there is a method in which a smear-stained specimen of a buffy coat in which leukocyte components in a blood sample are concentrated is examined. In general, the frequency of detection of bacteria in buffy coat specimens is about 30% in adult bacteremia, similar to the frequency of ear phlebotomy, but in newborns, bacteria are detected in 7 of 10 cases (70%). There is also a report, and information on the presence or absence of peripheral blood bacteria by microscopic examination of smears is a great guide for treatment.
Japanese Unexamined Patent Publication No. 60-224068 JP-T61-502376 Yoshino Ogino, "Acceleration of bacteria identification by gas chromatography", clinical examination, vol.29, No.12, pp.1618-1623, November 1985, Medical School

  In the above prior art, as the pretreatment operation, at least 1 to 2 days for selective separation of bacteria from the specimen, 1 day for enrichment, 1 day or more for fixation operation, a total of 3 to 4 days takes enough, In reality, this culture will continue until the bacteria grow, so the pretreatment operation often takes more than a week, and even when bacteria other than the causative bacteria of the disease are mixed, There is also.

  And importantly, because of the circumstances described above, many bacteria in the specimen to be cultured are taken up by phagocytes and are dead or quiescent due to antibiotic administration, so the number of bacteria that can grow under culture conditions. There are few, and the detection rate of actual bacteria by culture using clinical specimens is very low, around 10%. In other words, even if the blood of a patient suspected of having a clinically suspected Streptococcus pyogenes infection is further cultured for more than one day and night, after all, 90% of the blood is still not found. .

  Under these circumstances, detection of the causative Streptococcus pyogenes infection is clinically suspected despite the fact that it is currently required to determine the causative bacteria and select antibiotics accordingly. Treatment without waiting for the result, that is, the effective antibiotic is administered to the widest variety of bacteria without knowing the causative bacteria, and if there is no effect, another antibiotic is administered. It relies on a trial and error method of switching to material.

  Therefore, recently, immunology of Streptococcus pyogenes bacteria using techniques such as 1) latex agglutination, 2) coagulation, 3) enzyme immunoassay, 4) gold particle assay, 5) liposome immunoassay, etc. Rapid diagnostic methods have been developed to detect automatically. In these methods, C polysaccharides on the surface layer of Streptococcus pyogenes are extracted using nitrous acid or an enzyme, and this is detected as an antigen.

  However, the above-described immunological test methods have problems that the measurement results do not coincide with the results of the culture method, that is, they may show false positives or false negatives, and the procedure is complicated.

Furthermore, the characteristics of the diagnostic method utilizing an antigen-antibody reaction, A group antigen possessed bacteria other than S. pyogenes bacteria (e.g., Streptococcus Anjinosasu (Streptococcus Anginosus) such as cells) also will be detected, the species specificity There is a disadvantage of being inferior.

  On the other hand, diagnostic criteria have also been proposed for clinical diagnosis of fulminant group A streptococcal infections (JAMA, 269, 390-391, 1993), but this is not suitable for early diagnosis. .

  Thus, despite the fact that Streptococcus pyogenes infection is a disease that requires a quick and reliable diagnosis, the conventional diagnosis method has not been able to cope with it.

  The present invention has been completed in view of the above-mentioned problems in the technical field. The gist of the present invention is that it is specific to DNA or RNA possessed by infectious disease-causing bacteria, particularly Streptococcus pyogenes. It is to elucidate the base sequence of a gene part that is inherently possessed by a probe having a high reactivity and Streptococcus pyogenes contained in the probe.

  That is, with the probe of the present invention, for example, the DNA of a bacterium still maintained in a bacterium that has been taken up and destroyed by phagocytic cells can be detected significantly based on its specificity using a hybridization method. Thereby, the causative bacteria of the infectious disease can be detected quickly and reliably without culturing and growing the bacteria. In addition, if the primers are designed with reference to the base sequence information of these probes, the infectious disease-causing bacteria can be identified by DNA amplification by the PCR method without performing hybridization.

  In addition, if a probe used for hybridization is non-radioactive, for example, a biotinylated probe, it can be detected using an optical microscope even in a general laboratory without a radioisotope facility, and the detection operation can be performed quickly and easily. .

  By using the probe of the present invention, for example, the infectious disease-causing bacteria taken up by phagocytic cells can be directly detected without proliferating, and the bacteria can be quickly and accurately identified. That is, in the diagnosis using the probe of the present invention, bacteria can be identified with one sample, and the time required for the diagnosis is about 1 to 2 days from 3 to 4 days of the conventional method (the detection rate is low). The detection rate is much higher.

  Therefore, in addition to providing epoch-making guidelines for the treatment of Streptococcus pyogenes infection, effective treatment can be performed early on infectious disease patients, and as a result, a reduction in mortality is also expected.

  In addition, among the infectious disease-causing bacteria, in particular, the base sequence of probes that react specifically with DNA possessed by Streptococcus pyogenes bacteria has been clarified, making it possible to prepare these probes artificially. . Furthermore, using primers prepared using part of the analyzed base sequence information, the DNA of infectious disease-causing bacteria contained in clinical samples is amplified by the PCR method, which is useful for rapid diagnosis of causative bacteria. be able to.

  Furthermore, by comparing and referring to the base sequence of Genomic DNA contained in the clinical specimen and the base sequence analyzed by the present invention, it is possible to quickly identify the bacterial species causing infection.

  Examples of probes derived from Streptococcus pyogenes bacteria that cause infectious diseases are shown below.

DNA probe derived from Streptococcus pyogenes
(1) Preparation of DNA probe derived from Streptococcus pyogenes Clinical strain Streptococcus pyogenes is cultured overnight in BHI (Brain Heart Infusion) medium. Genomic DNA was extracted according to the Saito-Miura modification method ("Preparation of transforming deoxyribonucleic acid by phenol treatment", Biochem. Biophys. Acta vol. 72 , pp. 619-629 (1963)).

The extracted DNA is completely digested with the restriction enzyme Hind III, randomly cloned into the vector pGEM-3Z, and the clones obtained are specific to Streptococcus pyogenes, that is, specific to the DNA possessed by natural Streptococcus pyogenes Six types of probes containing DNA fragments that showed reactivity were selected.

  The selected probes are probe SP-6-28, probe SP-7-44, probe SP-14-1, probe SP-26-36, probe SP-26-46, and probe SP-55-3. Named.

(2) Examination of species specificity of DNA probe derived from Streptococcus pyogenes The reactivity of each probe with DNA of various infectious disease-causing strains was examined by the following method.

First, clinical isolates and deposited strains listed in Table 1 below were prepared as the strains to be examined. In Table 1, Escherichia coli K-12 and JM109 transformants containing leukocytes collected from 4 healthy adult males and plasmid pGEM-3Z were used as the sources of human genomic DNA and control samples. Prepared each.

そして、各臨床菌株を実施例１(1) に記載の方法に従って、各菌株が保有するＤＮＡを抽出し、この抽出したＤＮＡの一定量（例えば、10〜100 ng）をナイロンフィルターにスポットし、アルカリ変性したものをドット・ブロット・ハイブリダイゼーションの試料とした。なお、ヒト・ゲノミックＤＮＡ試料は、前出のSaito-Miura変法に、先に入手した白血球を適用することで調製した。一方、対照試料は、後述する実施例２(1) に記載のプラスミドＤＮＡの調製方法に、先に入手したプラスミド pGEM-3Zを含んだEscherichia coli K-12, JM109 形質転換体を適用することで調製した。次いで、Digoxigenin-11-dUTP (BRL社製) でラベルしたストレプトコッカス・ピオゲネス菌由来のＤＮＡプローブで、マニアティスのマニュアル（T. Maniatis,et al., "Molecular Cloning (A Laboratory Manual Second Edition)", Cold Spring Harbour Laboratory (1989))に従い、45％ホルムアミド、５×SSC、42℃の条件下で、終夜ハイブリダイゼーションを実施した。

Then, according to the method described in Example 1 (1), each clinical strain is extracted with DNA possessed by each strain, and a certain amount (for example, 10 to 100 ng) of the extracted DNA is spotted on a nylon filter. The alkali-denatured sample was used as a dot blot hybridization sample. The human genomic DNA sample was prepared by applying the previously obtained leukocytes to the modified Saito-Miura method described above. On the other hand, the control sample was obtained by applying the Escherichia coli K-12, JM109 transformant containing the previously obtained plasmid pGEM-3Z to the plasmid DNA preparation method described in Example 2 (1) described later. Prepared. Next, a DNA probe derived from Streptococcus pyogenes labeled with Digoxigenin-11-dUTP (manufactured by BRL), Maniatis manual (T. Maniatis, et al., "Molecular Cloning (A Laboratory Manual Second Edition)", According to Cold Spring Harbor Laboratory (1989)), hybridization was performed overnight under conditions of 45% formamide, 5 × SSC, and 42 ° C.

  Samples that had been hybridized overnight were washed twice with 0.1xSSC and 0.1% SDS at 55 ° C for 20 minutes according to the manual, and then detected and developed with Anti-Dig-ALP conjugates (BRL). And the status of hybridization was confirmed. The obtained results are as shown in FIG. Fig. 1 (a) shows the arrangement of strains of DNA spotted on each of the filters subjected to dot blot hybridization, and Fig. 1 (b) shows the above-mentioned probes SP-6-28 and SP. 7 shows the result after hybridization using -7-44, SP-14-1, SP-26-36, SP-26-46 and SP-55-3 to develop color.

  The experimental results regarding the reactivity between each probe and the DNA of each clinical strain are shown in Table 2 below.

上記図１および表２から明らかなように、いずれのプローブもストレプトコッカス・ピオゲネス菌に由来するＤＮＡに対してのみ反応性を示し、ストレプトコッカス属以外の菌種由来のＤＮＡはもちろん、ストレプトコッカス属の他の菌種由来のＤＮＡに対しても反応性（ハイブリッドの形成）が認められず、その種特異性が確認された。

As is clear from FIG. 1 and Table 2 above, all of the probes showed reactivity only with DNA derived from Streptococcus pyogenes, and DNAs derived from bacterial species other than Streptococcus were used as well as other Streptococcus species. Reactivity (hybrid formation) was not observed even for DNA derived from fungal species, and its species specificity was confirmed.

Base sequence analysis The base sequences of the DNA probes (6 in total) whose species specificity was confirmed in Example 1 were determined according to the following method.

(1) Preparation of plasmid DNA Escherichia coli K-12, JM109 transformant containing the subcloned insert (to determine the nucleotide sequence) in pGEM-3Z (Promega) was added to 5 ml of Luria-Bactani.
Medium (bacto-tryptone, 10g / 1L; bacto-yeast extract, 5g / 1L; NaCl, 10g / 1L; adjusted to pH 7.0 with 5N NaOH) and inoculated overnight.

The culture broth was collected by centrifugation (5,000 rpm, 5 min.). 50 mM glucose / 50 mM Tris-HCl (pH 8.0) / 10 mM containing lysozyme (Sigma) at a concentration of 2.5 mg / ml in the precipitate
100 μl of EDTA solution was added and left at room temperature for 5 minutes. To the obtained suspension, a 0.2 M aqueous sodium hydroxide solution containing sodium dodecyl sulfate (Sigma) at a concentration of 1% was added and mixed. Further, 150 μl of 5 M aqueous potassium acetate solution (pH 4.8) was added and mixed, and the mixture was ice-cooled for 15 minutes.

Then, the supernatant obtained by centrifugation (15,000 rpm, 15 min.) Is treated with phenol / CHCl ₃ , 2 times the amount of ethanol is added to the supernatant, and further centrifuged (12,000 rpm, 5 min.) To precipitate. Got. This precipitate was dissolved in 100 μl of 10 mM Tris-HCl (pH 7.5) /0.1 mM EDTA solution, 10 mg / ml RNase A (Sigma) solution was added, and the mixture was allowed to stand at room temperature for 15 minutes.

To this preparation, 300 μl of 0.1M aqueous sodium acetate solution (pH 4.8) was added, treated with phenol / CHCl ₃ , and ethanol was added to the supernatant to obtain a precipitate. This precipitate was dried and dissolved in 10 μl of distilled water to obtain a DNA sample.

(2) Pretreatment for base sequence determination Pretreatment for base sequence determination was performed using AutoRead (registered trademark) Sequencing Kit (Pharmacia).

  That is, the template DNA was adjusted to a concentration of 5 to 10 μg in a 32 μl solution. 32 μl of template DNA was transferred to a 1.5 ml minitube (Eppendorf), and 8 μl of 2M sodium hydroxide aqueous solution was added and gently mixed. Then, after light centrifugation, it was left at room temperature for 10 minutes.

  7 μl of 3M sodium acetate (pH 4.8) and 4 μl of distilled water were added, 120 μl of ethanol was further added and mixed, and the mixture was left on ethanol / dry ice for 15 minutes. Then, the precipitated DNA was collected by centrifugation for 15 minutes, and the supernatant was carefully removed. The resulting precipitate was washed with 70% ethanol and centrifuged for 10 minutes. Then, the supernatant was again removed with care, and the precipitate was dried under reduced pressure.

The precipitate was dissolved in 10 μl of distilled water, and fluorescent primer [Fluorescent Primer, Universal Primer; 5′-Fluorescein-d [CGACGTTGTAAAACGACGGCCAGT (SEQ ID NO: 7)]-3 ′ (1.6 pmol / μl; 0.42 A ₂₆₀ unit Reverse Primer, 5′-Fluorescein-d [CAGGAAACAGCTATGAC (SEQ ID NO: 8)]-3 ′ (2.1 pmol / μl; 0.42 A ₂₆₀ unit / ml)] 2 μl (0.42 A ₂₆₀ unit / ml, 4- 6 pmol) and 2 μl of annealing buffer were added and mixed gently.

Then, after light centrifugation, heat treatment was performed at 65 ° C. for 5 minutes, and quickly placed at 37 ° C., where the temperature was kept for 10 minutes. After incubating, the mixture was left at room temperature for 10 minutes or more and lightly centrifuged.
A sample obtained by adding 1 μl of extension buffer and 3 μl of dimethyl sulfoxide was used as a sample.

Fill A, C, and G and T in four mini tubes, AMIX to each tube (dATP the ddATP, dCTP, obtained by dissolving with c ⁷ dGTP and dTTP), dATP and C Mix (ddCTP, dCTP, c ⁷ dGTP and dTTP), G Mix (ddGTP dissolved with dATP, dCTP, c ⁷ dGTP and dTTP) and T Mix (ddTTP dissolved with dATP, dCTP, c ⁷ dGTP and dTTP) 2.5 μl was dispensed. Each solution was stored in ice until use, and kept at 37 ° C. for 1 minute or more before use.

2 μl of diluted T7 DNA polymerase (Pharmacia; 6-8 units / 2 μl) was added to the DNA sample and mixed thoroughly by pipetting or gentle mixing.
Immediately after mixing, this mixed solution was dispensed into 4 types of solutions kept at 4.5 μl each. A new tip was used for dispensing.

The mixture was incubated at 37 ° C. for 5 minutes, and 5 μl of the stop solution was added to each reaction solution.
A new tip was also used in this dispensing. The mixture was kept at 90 ° C. for 2 to 3 minutes and immediately cooled in ice. For electrophoresis, 4 to 6 μl was run per lane.

(3) Determination of base sequence Determination of the base sequence of each probe having specificity for DNA possessed by Streptococcus pyogenes disclosed in Examples 1 and 2 was performed at an electrophoresis temperature of 45 ° C and an electrophoresis time of 6 hours. The ALFDNA Sequencer system (Pharmacia) was used. Primers were sequentially designed from the sequences revealed from each upstream and downstream, and the above operation was repeated.

  As a result, probe SP-6-28 (SEQ ID NO: 1), probe SP-7-44 (SEQ ID NO: 2), probe SP-14-1 (SEQ ID NO: 3), probe SP-26-36 (sequence) No. 4), probe SP-26-46 (SEQ ID NO: 5), and probe SP-55-3 (SEQ ID NO: 6) were all revealed.

  As described above, the present invention not only provides a probe for diagnosing infectious diseases, which was the intended purpose, but also as a guideline for preparing PCR primers and for comparative reference with genomic DNA contained in clinical samples. Expected to have excellent usefulness as a suitable standard sequence, and also provides excellent effects such as providing valuable clues in the future research and development of probes that specifically react with DNA possessed by infectious disease-causing bacteria. Is.

  The base sequence disclosed in the present application is obtained by random cloning of genomic DNA of clinical isolates. Therefore, the usefulness of the base sequence of the present invention extends to its complementary strand. It is.

  Furthermore, it is naturally considered that there is a mutated portion in the DNA possessed by the wild strain, but as is apparent from the disclosure of the above examples, the DNA mutated portion is used for hybridization for infectious disease diagnosis. The usefulness of the present invention, such as the specificity of the probe of the present invention, or the use of the nucleotide sequence information disclosed in the present application for designing primers of PCR methods for rapid diagnosis of infectious diseases, etc. It has no effect.

(a) shows the arrangement of DNA strains in each dot-blot hybridization filter, and (b) shows the result after hybridization with each probe for color development.

Claims (1)

Streptococcus pyogenes , which is a causative bacterium of an infectious disease, contains a DNA fragment having the base sequence of SEQ ID NO: 3, which can be obtained by treating DNA possessed by Streptococcus pyogenes with restriction enzyme HindIII, and Streptococcus pyogenes A probe for diagnosing infectious diseases, characterized in that it specifically hybridizes with DNA possessed by.

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