US20030064388A1

US20030064388A1 - Oligonucleotides for detecting bacteria and detection process

Info

Publication number: US20030064388A1
Application number: US10/138,381
Authority: US
Inventors: Tomoko Nakayama; Jun Tada; Shigeru Fukushima; Tetsuo Ohashi
Original assignee: Shimadzu Corp
Current assignee: Shimadzu Corp
Priority date: 1994-02-28
Filing date: 2002-05-06
Publication date: 2003-04-03
Also published as: EP0669399B1; US6218110B1; EP1036846A2; EP0669399A3; DE69433201D1; EP0669399A2; DE69433201T2; US5795717A; EP1036846A3

Abstract

A synthetic oligonucleotide which is complementary to a nucleotide sequence of a gene selected from the group consisting of the Shiga toxin gene of Shigella species, the ipaH gene of Shigella species and EIEC, the invE gene of Shigella species and EIEC, the araC gene of Salmonella species, the Verocytotoxin-1 gene of EHEC or VTEC, the Verocytotoxin-2 gene of EHEC or VTEC, the toxic shock syndrome toxin-1 gene of Staphylococcus aureus, the ctx gene of Vibrio cholerae, and the enterotoxin gene of Clostridium perfringens; a method for detecting a bacterial strain by amplifying a region of the above gene by PCR using the above oligonucleotides as primers and detecting the amplified region; and a kit for the detection of the bacterial strain.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to detection of pathogenic bacteria in samples (e.g., clinical isolates and food specimens) for the purposes of diagnoses, screenings, quarantine inspections, and clinical tests. Specifically, it relates to detection of pathogens associated with bacterial food poisoning and bacterial diarrhea. More specifically, it relates to detection of enteropathogenic bacteria including Shigella species, Salmonella species, enterohemorrhagic Escherichia coli or Verocytotoxin-producing Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Clostridlum perfringens.

2. Discussion of the Related Art

Detection of pathogenic bacteria such as Shigella species, Salmonella species, enterohemorrhagic Escherichia coli (hereinafter simply referred to as EHEC) or Verocytotoxin-producing Escherichia coli (hereinafter simply referred to as VTEC), Staphylococcus aureus, Vibrio cholerae, and Clostridium perfringens is an important task in the field of medicine and public hygiene, and various methods have been used.

Conventionally, detection of a pathogenic bacterial strain involves isolation of several pathogenic bacterial colonies and identification of the species of the bacteria by serological or biochemical method.

In the case of Shigella species, this has been achieved by culturing and isolating the target bacterium from specimens of patient stools, food, or the like, using a medium, such as DHL agar or MacConkey's agar, and then further culturing the bacterium using a medium such as TSI agar or LIM agar for the purpose of identification.

In the case of Salmonella species, culture is conducted for isolation of the bacteria from specimens of patient stools or vomits, food or wiping samples, etc., followed by inoculation to TSI agar, SIM medium, VP-MR medium and lysine decarboxylation test medium and subsequent overnight culture at 37° C., to confirm Salmonella species, and the serotype is determined using a commercially available set of antisera against O and H antigens,

EHEC or VTEC has been found to cause hemolytic uremic syndrome in children, as well as food poisoning symptoms, typically hemorrhagic colitis, and stress has recently been placed on detection of this bacterium in clinical tests. In the case of detecting EHEC or VTEC, specimens are patient stools, food, or water samples (drinking water, river water, etc.) collected from the environment surrounding the patient. In detecting EHEC (VTEC) in these specimens, it is necessary to perform a series of procedures from direct isolation culture, a primary confirmation culture test, and a secondary confirmation culture test to an agglutination test with an antiserum.

In the case of Staphylococcus aureus, specimens are patient vomits or stools, food the patient ate, samples wiped out from the environment surrounding the patient, or the like. Before Staphylococcus aureus is detected and identified in these specimens, it is necessary to perform bacterial culture, isolation culture and then pure culture and confirmation culture.

In the case of Vibrio cholerae, specimens are patient stools or food, or water samples (drinking water, river water, sea water, etc.) or benthos samples collected from the environment surrounding the patient. In detecting and identifying Vibrio cholerae in these specimens, it is necessary to perform a series of procedures from primary enrichment culture, secondary enrichment culture, and isolation culture to an agglutination reaction test with anti-V. cholerae 01 serum and confirmation culture.

In the case of Clostridium perfringens, specimens are obtained mainly from patient stools and food. For detection and identification, the specimens are subjected to enrichment culture and isolation culture under anaerobic conditions. With several colonies of the bacteria, tests for biochemical properties are conducted.

Any identification process mentioned above usually takes several days, and hampers rapid diagnoses of infectious diseases.

Specifically, in the case of Shigella species, each culture step takes 18-24 hours, totalling 3-4 days; rapid detection is difficult. Other available methods include the reversed passive latex agglutination using a specific antibody to the Shiga toxin, the EIA method using a specific antibody to the 140 MDal plasmid product associated with the pathogenicity of Shigella species and enteroinvasive Escherishia coli [Kenichiro Ito et al., Japanese Journal of Bacteriology 41, 414 (1986)] and the DNA probe method for detecting the ipaB gene, the ipac gene, or the ipaD gene (U.S. patent application Ser. No. 888194).

However, these testing methods require complicated troublesome procedures in preparing reagents and specimens, and take much time.

In the case of Salmonella species, 2-3 days are taken for bacterial isolation and identification of the bacteria from specimens. In addition, Salmonella tests are difficult to conduct in ordinary laboratories, because as many as 100 antisera and much experience are required to achieve complete serum typing of Salmonella species, which involve a large number of serum types. Also, each culture step and serotyping test take 3-4 days; rapidity is poor. Moreover, confirmation culture and serotyping are expensive and involve troublesome operation.

In the case of EHEC (VTEC), each culture step takes 18-24 hours, totalling as many as 3-4 days. The currently representative serotype of EHEC (VTEC) is 0157:H7, but no diagnostic antiserum has been commercially available for identification of this serotype, so that the diagnostic antiserum has to be prepared by the investigator. In addition, it is often difficult to identify the causative bacterium solely on the basis of serum typing in EHEC (VTEC), because the serum type and the pathogenicity do not always agree with each other. Therefore, the conventional testing method for EHEC (VTEC) lacks rapidity and simplicity, and is not suitable for practical application.

In the case of Staphylococcus aureus, each culture step takes 18-24 hours, totalling as many as about 4 days when combined with the time required for the subsequent testings. Also, in the biochemical test in culture for identification, various properties, such as aerobic growth, VP reactivity, nitrate reduction, Tween 80 hydrolyzability, hyaluronidase activity and sugar decomposition, should be examined, but this process is troublesome, tedious and expensive. The most reliable method for identifying the causative bacterium for food poisoning and diarrhea is to test the isolated strain for exotoxin (toxic shock syndrome toxin -1, hereinafter simply referred to as TSST-1) production. However, even when a commercially available convenient reagent kit is used, 18-20 hours will be taken to obtain the results; rapidity is poor.

In the case of Vibrio cholerae, each culture step takes 18-24 hours, totalling as many as about 4 days. In the biochemical test concerning confirmation culture, various properties, such as oxidase test positivity, indole test positivity, motility, and lysine decarboxylation test positivity should be examined. These tests are troublesome, tedious and expensive, and the results obtained are difficult to assess in some cases. Moreover, in the case of Vibrio cholerae, it is essential to test the isolated strain for enterotoxin (cholera toxin; CT) production to take an administrative measure for pest control. However, even when a commercially available convenient reagent kit is used, 18-20 hours will be taken to obtain the results; rapidity is poor and practical applicability is low.

In the case of Welch's bacillus( Clostridium perfringens), the detection requires considerably long time: each culture step takes 18-48 hours, totalling 5-6 days. In addition, since Clostridium perfringens strains are widely distributed in the nature, only the detection of the bacterial strain from specimens is not enough to determine the strain as the causative agent for food poisoning. Further tests are required, including detection of the enterotoxin in patient stool, assay of the isolated strain for enterotoxin production, serotype determination, and bacterial count for suspected food.

These procedures consume much time and labor, and lack rapidity and simplicity.

In recent years, the DNA probing or hybridization using oligonucleotides has been tried. However, when hybridization is performed on a membrane or on other supports using a probe of a labeled oligonucleotide, followed by detection of the probe, sensitivity of the assays depends on numbers of organisms available for detection. Therefore it is difficult to achieve a high detection sensitivity and selectivity in this test without the above-described pretreatment of the separation culture.

SUMMARY OF THE INVENTION

It is object of the present invention to provide synthetic oligonucleotides used as primers for PCR to amplify certain regions of the genes specific to the above various pathogenic microorganisums.

It is another object of the present invention to provide a simple, rapid and highly sensitive process for detecting the above various pathogenic microorganisms for quarantine inspection, clinical laboratory examination and food inspection, wherein a region of a gene specific to the bacterial strain to be detected is amplified by the PCR technique using synthetic oligonucleotide primers.

It is still another object of the present invention to provide a kit for detection of the above various bacterial strains, comprising at least a pair of primers, a thermostable DNA polymerase, and dNTP solutions.

The gist of the present invention relates to:

1) A synthetic oligonucleotide of 10 to 30 bases which is complementary to a nucleotide sequence of a gene selected from the group consisting of the Shiga toxin gene of Shigella species, the ipaH gene of Shigella species and enteroinvasive Escherishia coli (hereinafter simply referred to as EIEC), the invE gene of Shigella species and EIEC, the araC gene of Salmonella species, the Verocytotoxin-1 gene of EHEC or VTEC, the Verocytotoxin-2 gene of EHEC or VTEC, the toxic shock syndrome toxin gene of Staphylococcus aureus, the ctx gene of Vibrio cholerae, and the enterotoxin gene of Clostridium perfringens;

2) A synthetic oligonucleotide comprising a nucleotide sequence complementary to the synthetic oligonucleotide of 1);

3) A method for detecting a bacterial strain selected from the group consisting of Shigella species, EIEC, Salmonella species, EHEC, VTEC, Staphylococcus aureus, Vibrio cholerae and Clostridium perfringens, wherein the method comprises:

(1) hybridizing one primer to a single-stranded target DNA as a template DNA present in a specimen and carrying out a primer extension reaction to give a primer extension product,

(2) denaturing the resulting DNA duplex to separate the primer extension product from the template DNA; the primer extension product functioning as the other template DNA for the other primer,

(3) repeating a cycle of simultaneous primer extension reaction with the two primers, separation of the primer extension products from the templates, and hybridization of primers to amplify a region of the target DNA, in the steps from (1) to (3), the primers being selected from the group consisting of oligonucleotides of 1) and 2),

(4) detecting the amplified nucleotide sequence to determine whether a suspected bacterial strain is present in the specimen; and

4) A kit for detection of a bacterial strain, comprising at least a pair of primers selected from the group consisting of oligonucleotides of 1) and 2), a thermostable DNA polymerase, and dNTP solutions.

The present invention provides a highly selective and highly sensitive method for rapid detection of Shigella species having the Shiga toxin gene, the ipaH gene and the invE gene, EIEC having the ipah gene and the invE gene, Salmonella species having the araC gene, EHEC having the VT1 gene and the VT2 gene, Staphylococcus aureus having the TSST-1 gene, Vibrio cholerae having the ctx gene, and Clostridium perfringens having the enterotoxin gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only, and thus, are not limitative of the present invention. [0035]
FIG. 1 is the pattern of the electrophoresis of the amplified DNA fragments on an agarose gel to evaluate the sensitivity of the detection method of the present invention for the TSST-1 gene of [0036] Staphylococcus aureus, wherein numerals 1 to 9 refer to the number of DNA copies used in the polymerase chain reaction (hereinafter simply referred to as PCR): 1 means 10⁷copies; 2, 10⁶copies; 3, 10⁵copies; 4, 10⁴copies; 5, 10³copies; 6, 10²copies; 7, 10 copies; 8, 1 copy; and 9, no DNA.
FIG. 2 is the pattern of the agarose gel electrophoresis of the nucleotide fragments amplified by PCR to evaluate the specificity of the detection method of the present invention for the ctx gene of [0037] Vibrio cholerae, wherein M indicates the molecular weight marker and lanes 1-13 indicate the template DNA solutions containing heat extract of the following strains:
[0038] Lanes 1 to 3: Vibrio cholerae (El Tor—Ogawa type, the ctx gene positive strain)
[0039] Lanes 4 to 6: Vibrio cholerae (El Tor—Inaba type, the ctx gene positive strain)
Lane 7: [0040] Vibrio cholerae (Classical—Ogawa type, the ctx gene positive strain)
Lane 8: [0041] Vibrio cholerae (Classical—Inaba type, the ctx gene positive strain)
[0042] Lanes 9 to 10: Vibrio cholerae (non-01, the ctx gene positive strain)
Lane 11: [0043] Vibrio cholerae (El Tor—Ogawa type, the ctx gene negative strain)
Lane 12: [0044] Vibrio cholerae (El Tor—Inaba type, the ctx gene negative strain)
Lane 13: Enterotoxigenic [0045] Escherichia coli(Thermolabile enterotoxin gene positive strain).
FIG. 3 is the electrophoretic pattern of the agarose gel electrophoresis for the nucleotide fragments amplified by PCR to detect the enterotoxin gene of [0046] Clostridium perfringens, the upper part being the results obtained with Oligonucleotide SEQ ID NO: 28+Oligonucleotide SEQ ID NO: 33; the lower part being the results obtained with Oligonucleotide SEQ ID NO: 29+Oligonucleotide SEQ ID NO: 33, wherein M indicates the molecular weight marker and lanes 1-13 indicate the template DNA derived from heat extracts of the following strains: Lane 1, ATCC 12925; Lane 2, ATCC 12924; Lane 3, ATCC 12922; Lane 4, ATCC 12920; Lane 5, ATCC 12916; Lane 6, ATCC 12915; Lane 7, ATCC 12918; Lane 8, ATCC 12919; Lane 9, ATCC 12921; Lane 10, JCM 1296; Lane 11, JCM 1416; Lane 12, JCM 1382; and Lane 13, negative control.
FIG. 4 shows the results of a Southern blot hybridization test to confirm if the nucleotide sequence of amplified DNA with the primers of the present invention is a part of the enterotoxin gene sequences of [0047] Clostridium perfringens, the upper part being the results obtained with Oligonucleotide SEQ ID NO: 28+Oligonucleotide SEQ ID NO: 33; the lower part being the results obtained with Oligonucleotide SEQ ID NO: 29+Oligonucleotide SEQ ID NO: 33, wherein M indicates the molecular weight marker and lanes 1-13 indicate the template DNA solutions derived from heat extracts of the following strains: Lane 1, ATCC 12925; Lane 2, ATCC 12924; Lane 3, ATCC 12922; Lane 4, ATCC 12920; Lane 5, ATCC 12916; Lane 6, ATCC 12915; Lane 7, ATCC 12918; Lane 8, ATCC 12919; Lane 9, ATCC 12921; Lane 10, JCM 1296; Lane 11, JCM 1416; Lane 12, JCM 1382; and Lane 13, negative control.

DETAILED DESCRIPTION OF THE INVENTION

Oligonucleotides [0048]
An oligonucleotide of the present invention is a synthetic oligonucleotide which complementarily and selectively hybridizes to a gene specific to a pathogenic bacterial strain such as Shigella species, EIEC, Salmonella species, EHEC or VTEC, [0049] Staphylococcus aureus, Vibrio cholerae, and Clostridium perfringens. In the bacterial detection of the present invention, the oligonucleotides are used as primers in the PCR to amplify a DNA sequence of a target gene specific to the pathogenic bacteria to be detected. For this purpose, any combination of two oligonucleotides of the present invention may optionally be employed without particular limitation as long as a DNA sequence of 50 to 2000 bases, preferably of 100 to 1000 bases, can be amplified. The preferred combination of oligonucleotides are described in detail in the following preferred embodiments.
In the oligonucleotides mentioned below, T may be replaced with U without impairing the advantageous effect of the oligonucleotide. [0050]
Preferred [0051] Embodiment 1
For the detection of Shigella species ([0052] Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnel) and EIEC, the Shiga toxin gene specific to Shigella species, the ipaH gene specific to Shigella species and EIEC, or the invE gene specific to Shigella species and EIEC is selected as the target gene.
When the Shiga toxin gene is targeted, two oligonucleotides, one comprising at least 10 consecutive bases of the following oligonucleotide SEQ ID No. 1 and the other comprising at least 10 consecutive bases of the following oligonucleotide SEQ ID NO: 2, are selected in the present invention: [0053]
(5′)-CAACACTGGATGATCTCAG-(3′) (SEQ ID NO: 1) [0054]
(5′)-CCCCCTCAACTGCTAATA-(3′) (SEQ ID NO: 2) [0055]
When the ipaH gene is targeted, two oligonucleotides, one comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 3 and the other comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 4 are selected in the present invention: [0056]
(5′)-TGTATCACAGATATGGCATGC-(3′) (SEQ ID NO: 3) [0057]
(5′)-TCCGGAGATTGTTCCATGTG-(3′) (SEQ ID NO: 4) [0058]
When the invE gene is targeted, two oligonucleotides, one comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 5 and the other comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 6 are selected in the present invention: [0059]
(5′)-CAAGATTTAACCTTCGTCAACC-(3′) (SEQ ID NO: 5) [0060]
(5′)-AGTTCTCGGATGCTATGCTC-(3′) (SEQ ID NO: 6) [0061]
Preferred [0062] Embodiment 2
For the detection of Salmonella species, the araC gene is targeted. [0063]
For this purpose, any one of the following oligonucleotide combinations is preferably selected in the present invention: [0064]
a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 7 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 8; a combitnation in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 9 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 10; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 11 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 8: [0065]
(5′)-CGCGGAGAGGGCGTCATT-(3′) (SEQ ID NO: 7) [0066]
(5′)-GCAACGACTCATTAATTACCG-(3′) (SEQ ID NO: 8) [0067]
(5′)-ATCTGGTCGCCGGGCTGA-(3′) (SEQ ID NO: 9) [0068]
(5′)-GCATCGCGCACACGGCTA-(3′) (SEQ ID NO: 10) [0069]
(5′)-GGCGAGCAGTTTGTCTGTC-(3′) (SEQ ID NO: 11) [0070]
Preferred [0071] Embodiment 3
For the detection of EHEC or VTEC strains, the Verocytotoxin-1 (VT1) gene or the Verocytotoxin-2 (VT2) gene is targeted. [0072]
In order to detect bacteria which have only the VT1 gene, two oligonucleotides, one comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 12 and the other comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 13, are preferably selected in the present invention: [0073]
(5′)-CAACACTGGATGATCTCAG-(3′) (SEQ ID NO: 12) [0074]
(5′)-CCCCCTCAACTGCTAATA-(3′) (SEQ ID NO: 13) [0075]
In order to detect bacteria which have only the VT2 gene or its variant genes (VT2vha, VT2vhb and VT2vp1), two oligonucleotides, one comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 14 and the other comprising at least 10 consecutive bases of oligonucleotide SEQ ID NO: 15, are preferably selected in the present invention: [0076]
(5′l)-ATCAGTCGTCACTCACTGGT-(31) (SEQ ID NO: 14) [0077]
(5′)-CCAGTTATCTGACATTCTG-(3′) (SEQ ID NO: 15) [0078]
In order to detect bacteria which have both the VT1 gene and the VT2 gene (including the VT2vha, VT2vhb and VT2vp1 genes), any one of the following oligonucleotide combinations is preferably selected in the present invention: [0079]
a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 16 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 18; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 17 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 18: [0080]
(5′)-AGTTTACGTTAGACTTTTCGAC-(3′) (SEQ ID NO: 16) [0081]
(5′)-CGGACAGTAGTTATACCAC-(3′) (SEQ ID NO: 17) [0082]
(5′)-CTGCTGTCACAGTGACAAA-(3′) (SEQ ID NO: 18) [0083]
Preferred [0084] Embodiment 4
For the detection of [0085] Staphylococcus aureus, the TSST-1 gene is targeted.
For this purpose, any one of the following oligonucleotide combination is preferably selected in the present invention: [0086]
a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 20 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 21; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 19 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 22; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 20 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 22: [0087]
(5′)-CCTTTAAAAGTTAAGGTTCATG-(3′) (SEQ ID NO: 19) [0088]
(5′)-GGCCAAAGTTCGATAAAAAAC-(3′) (SEQ ID NO: 20) [0089]
(5′)-ATTTATAGGTGGTTTTTCAGTAT-(3′) (SEQ ID NO: 21) [0090]
(5′)-CTGCTTCTATAGTTTTTATTTCA-(3′) (SEQ ID NO: 22) [0091]
Preferred [0092] Embodiment 5
For the detection of [0093] Vibrio cholerae, the ctx gene is targeted.
For this purpose, any one of the following oligonucleotide combinations is preferably selected in the present invention: [0094]
a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 23 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 25; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 24 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 26: [0095]
(5′)-TGATGAAATAAAGCAGTCAGGT-(3′) (SEQ ID NO: 23) [0096]
(5′)-ACAGAGTGAGTACTTTGACC-(3′) (SEQ ID NO: 24) [0097]
(5′)-GGCACTTCTCAAACTAATTGAG-(3′) (SEQ ID NO: 25) [0098]
(5′)-ATACCATCCATATATTTGGGAG-(3′) (SEQ ID NO: 26) [0099]
Preferred [0100] Embodiment 6
For the detection of [0101] Clostridium perfringens, the enterotoxin gene is targeted.
For this purpose, any one of the following oligonucleotide combinations is preferably selected in the present invention:[0102]
a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 27 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 32; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 28 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 33; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 29 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 33; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 30 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 34; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 31 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 35: [0103]
(5′)-TCTGAGGATTTAAAAACACC-(3′) (SEQ ID NO: 27) [0104]
(5′)-ACCCTCAGTAGGTTCAAGTC-(3′) (SEQ ID NO: 28) [0105]
(5′)-ATGAAACAGGTACCTTTAGCC-(3′) (SEQ ID NO: 29) [0106]
(5′)-GGTAATATCTCTGATGATGGAT-(3′) (SEQ ID NO: 30) [0107]
(5′)-TAACTCATACCCTTGGACTC-(3′) (SEQ ID NO: 31) [0108]
(5′)-GAACCTTGATCAATATTTCC-(3′) (SEQ ID NO: 32) [0109]
(5′)-GTAGCAGCAGCTAAATCAAGG-(3′) (SEQ ID NO: 33) [0110]
(5′)-AGTCCAAGGGTATGAGTTAG-(3′) (SEQ ID NO: 34) [0111]
(5′)-CCATCACCTAAGGACTGTTC-(3′) (SEQ ID NO: 35) [0112]
Amplification of Gene Sequence by PCR [0113]
For amplification of a region of a target gene in the present invention, the PCR developed by Saiki et al. [Science 230, 1350 (1985)] is employed. [0114]
Specifically, two oligonucleotide primers that flank a specific region of a target gene (in the present invention, the Shiga toxin gene of Shigella species, the ipaH and invE genes of Shigella species and EIEC, the araC gene of Salmonella species, the VT1 and VT2 genes of EHEC or VTEC, the TSST-1 gene of [0115] Staphylococcus aureus, the ctx gene of Vibrio cholerae and the enterotoxin gene of Clostridium perfringens) are synthesized.
In PCR, one of the oligonucleotide primers selectively hybridizes to the (+)-strand of a target gene DNA, and the other hybridizes to the (−)-strand of the DNA. Then, both the oligonucleotides serve as primers of template dependent DNA polymerization respectively. In the present invention, single strand DNAs formed by heat denaturation of double strand DNAs in specimens are used as templates. The duplexes resulting from the DNA polymerization reaction are then denatured to separate the primer extension products from the templates. Then, the primer extension products themselves serve as the templates for the next DNA polymerization reaction. The cycle of denaturation, primer annealing in which a primer hybridizes with a template DNA and a primer extension reaction is repeated until the region of the target gene is amplified enough for its detection. [0116]
Specimens applicable to the PCR in the present invention may include clinical samples such as stool, urine, blood, tissue homogenate, and food samples. A specimen for PCR should be pre-treated to release the nucleic acid components from the bacterial cells present therein. Since PCR can be carried out with only several to several tens of nucleic acid molecules, a test solution containing an adequate amount of nucleic acid can be prepared simply by treating a specimen with a bacteriolytic enzyme, a surfactant or an alkali for a short time. [0117]
Oligonucleotides used as primers in the present invention may be either synthetic or natural, and in view of selectivity, detection sensitivity and reproducibility, they are not less than 10 bases in length, preferably not less than 15 bases. It is not necessary to label the primers for detection. [0118]
The region to be amplified in a target gene (i.e., the Shiga toxin gene of Shigella species, the ipah gene and the invE gene of EIEC, the VT1 gene and the VT2 gene of EHEC or VTEC, the arac gene of Salmonella species, the TSST-1 gene of [0119] Staphylococcus aureus, and the ctx gene of Vibrio cholerae, and the enterotoxin gene of Clostridium perfringens) is 50 to 2000 bases in length, preferably 100 to 1000 bases.
In PCR, a thermostable DNA polymerase is used. The origins from which the enzyme is derived are not particularly limited as long as the enzyme maintains its activity at a temperature of from 90 to 95° C. The denaturation is carried out at a temperature of from 90 to 95° C., the primer annealing from 37 to 65° C., and the polymerization reaction from 50 to 75° C. The cycle of denaturation, primer annealing and polymerization is repeated for 20 to 42 cycles. [0120]
The presence or absence, and the length of the amplified nucleotide fragment can be detected by subjecting the reaction solution to agarose gel electrophoresis after the completion of PCR. Other types of electrophoresis and chromatography can also be used for the detection. One of the oligonucleotide primers may be used as a probe to detect the amplified nucleotide sequence. [0121]
The detection of a nucleotide sequence of a target gene in a specimen means that the bacterial strain having the gene is present in the specimen. [0122]
The invention will now be described in more detail by the following examples, but it should be noted that the invention is not limited to these examples. [0123]

EXAMPLES

Example 1

Detection of Shigella Species having the Shiga Toxin Gene

[Experiment 1][0124]
Preparation of Specimens [0125]
The 42 strains of [0126] Shigella dysenteriae listed in Table 1 are obtained from patients or other sources. Each strain is inoculated to LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride), and subjected to overnight shaking culture at 37° C. under aerobic conditions. Each culture broth is diluted 10 folds with 10 mM Tris-HCl buffer, pH 7.5 (hereinafter referred to as TE buffer), and heated at 95° C. for 10 minutes, followed by centrifugation; the supernatants are used as specimen solutions.
Synthesis of Primers [0127]
As primers for amplifying the Shiga toxin gene of [0128] Shigella dysenteriae, the above-described oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 are selected based upon the known base sequence of the Shiga toxin gene [Takao, T. et al., Microb. Pathog., 5:357-369 (1988)], and chemically synthesized by the β-cyanoethylphosphoamidite method using a Cyclone Plus DNA synthesizer (produced by MilliGen/Bioresearch). The synthesized oligonucleotides are purified by high performance liquid chromatography using a C18 reversed-phase column.
The Shiga toxin gene is regarded as identical to the VT1 gene of EHEC or VTEC, with difference only in several bases [Takao, T. et al., Microb. Pathog., 5:357-369 (1988)]. [0129]
PCR [0130]
To 3 μl of the above-described specimen solution, 17.05 μl of sterile distilled water, 3 μl of 10×reaction buffer, 4.8 μl of dNTP solution, 1.0 μl of primer (1), 1.0 μl of primer (2), and 0.15 μl of a thermostable DNA polymerase are added to prepare 30 μl of a reaction mixture. This reaction mixture is overlaid with 50 μl of mineral oil (produced by SIGMA). The contents of the solutions used and the primers (1) and (2) are as follows: [0131]
10×reaction buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 15 mM MgCl[0132] ₂, 0.1% (w/v) gelatin.
dNTP solution: A mixture of dATP, dCTP, dGTP and dTTP, each having a final concentration of 1.25 mM. [0133]
Primers (1) and (2): Aqueous solution of the above-described chemically synthesized purified products (concentration, 3.75 OD/ml). [0134]
Primers: The above-described chemically synthesized and purified products are used in combination as follows:[0135]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 1+Oligonucleotide SEQ ID NO: 2 [0136]
Thermostable DNA polymerase: Taq DNA polymerase (5 unit/ml; produced by Perkin Elmer Cetus). [0137]
The reaction conditions are as follows:[0138]
Thermal denaturation: 94° C. for 1 minute. [0139]
Annealing: 55° C. for 1 minute. [0140]
Polymerization: 72° C. for 1 minute.[0141]
The cycle of thermal denaturation, primer annealing and polymerization (5.7 minutes) is repeated for 35 cycles (entire time, about 3 hours). This procedure is performed using a DNA thermal cycler (produced by Perkin Elmer Cetus) in which the above reaction conditions are programmed. [0142]
Detection [0143]
Agarose Gel Electrophoresis [0144]
To detect the amplified nucleotide fragment in the reaction mixture, agarose gel electrophoresis is conducted as mentioned below. [0145]
The agarose gel used has a gel concentration of 3% (w/v) and contains ethidium bromide (0.5 μl/ml). Electrophoresis is performed at the constant voltage of 100 V for 30 minutes. Operation procedures and other conditions described by Maniatis et al.[Molecular Cloning, 2nd edition (1989)] are used. In addition to the reaction mixture, molecular weight markers are also electrophoresed concurrently. The length of the nucleotide fragment is calculated by comparing the relative mobilities. [0146]
Reversed Passive Latex Agglutination (RPLA) Test [0147]
A commercially available RPLA kit for detection of [0148] Escherichia coli Verocytotoxin (produced by DENKA SEIKEN) is purchased. Specimens are prepared and tested according to the instruction manual attached.
Results [0149]

The base sequence of the Shiga toxin gene of Shigella dysenteriae has already been determined. Therefore, the length of the nucleotide amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when the oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention are used in combination, a nucleotide sequence of 349 bases (or a nucleotide duplex of 349 base pairs) is amplified. When this estimation accords with the length of the amplified nucleotide fragment, it is judged that PCR using the combination of primers accurately amplify the target region of the Shiga toxin gene, and that the bacterial strain in the specimen has the Shiga toxin gene. The results obtained from the agarose gel electrophoresis with 34 test strains are given in Table 1. PCR using the primers of the present invention amplifies only the DNA of the strains which give positive results in the RPLA, showing no amplification of DNAs of Shiga toxin negative strains. This indicates that PCR using the primers of the present invention are capable of accurately amplifying the Shiga toxin gene and that Shigella dysenteriae having the Shiga toxin gene can be detected with high accuracy by using the oligonucleotides of the present invention.

TABLE 1


No	Strains	RPLA	PCR

01	S. dysenteriae	TUMD 1	−	−
02	S. dysenteriae	TUMD 2	−	−
03	S. dysenteriae	TUMD 3	−	−
04	S. dysenteriae	TUMD 4	−	−
05	S. dysenteriae	TUMD 5	−	−
06	S. dysenteriae	TUMD 6	−	−
07	S. dysenteriae	MARABLA	−	−
08	S. dysenteriae	AQ7003	+	+
09	S. dysenteriae	AQ7004	+	+
10	S. dysenteriae	AQ7018	−	−
11	S. dysenteriae	AQ7029	−	−
12	S. dysenteriae	AQ7030	−	−
13	S. dysenteriae	AQ7061	−	−
14	S. dysenteriae	AQ7125	−	−
15	S. dysenteriae	AQ7131	−	−
16	S. dysenteriae	AQ7151	−	−
17	S. dysenteriae	AQ7164	−	−
18	S. dysenteriae	AQ7166	−	−
19	S. dysenteriae	AQ7234	−	−
20	S. dysenteriae	AQ7302	−	−
21	S. dysenteriae	AQ7350	−	−
22	S. dysenteriae	AQ7370	−	−
23	S. dysenteriae	AQ7403	−	−
24	S. dysenteriae	AA-22021	+	+
25	S. dysenteriae	AA-22184	+	+
26	S. dysenteriae	AA-22192	+	+
27	S. dysenteriae	AA-22555	+	+
28	S. dysenteriae	AA-21933	+	+
29	S. dysenteriae	AA-22496	+	+
30	S. dysenteriae	AA-22224	+	+
31	S. dysenteriae	AA-22542	+	+
32	S. dysenteriae	AA-22616	+	+
33	S. dysenteriae	AA-22239	+	+
34	S. dysenteriae	AA-22538	+	+
35	S. dysenteriae	ATCC9361	+	+
36	S. dysenteriae	ATCC9753	−	−
37	S. dysenteriae	ATCC9764	−	−
38	S. dysenteriae	ATCC11456a	+	+
39	S. dysenteriae	ATCC13313	+	+
40	S. dysenteriae	ATCC23351	+	+
41	S. dysenteriae	ATCC29027	−	−
42	S. dysenteriae	ATCC29028	−	−

[Experiment 2][0151]
To determine whether the results obtained in [0152] Experiment 1 are specific to the Shiga toxin gene, the DNAs of clinically important pathogenic bacteria other than Shigella dysenteriae are examined with the primers of the present invention. The same procedure as used in Experiment 1 is followed, except for the method of preparation of specimens.
Preparation of Specimens [0153]
Each strain listed in Table 2 is inoculated to an appropriate enrichment medium, and subjected to overnight culture at 37° C. under aerobic or anaerobic conditions ([0154] Clostridium perfringens, Campylobacter jejuni, Campylobacter coli, Bacteroides flagilis, Bacteroides vulgatus, Lactobacillus acidophilus, and Bifidobacterium adolescentis are cultured under anaerobic conditions, while Neisseria gonorrhoeae and Neisseria meningitidis are cultured in the presence of 3-10% CO₂). Bacterial cells are centrifugally recovered from 0.5 ml of each culture broth, and once washed with TE buffer. To these bacterial cells, an N-acetylmuraminidase solution in 50 mM phosphate buffer, pH 7.5, and an achromopeptidase solution in the same buffer are added to final concentrations of 50 μg/ml and 1 mg/ml, respectively, followed by incubation at 37° C. for 10 minutes to lyse the cells. A 1:1 phenol/chloroform mixture, saturated with TE buffer, is added to the lysate, followed by vigorous stirring. After centrifugation, the supernatant is recovered, and treated with ethanol to precipitate the nucleic acids. The resulting precipitate is dissolved in 1 ml of TE buffer; this solution is used as a specimen. Also, Human placenta DNA, at a concentration of 1 μg/ml, is subjected to PCR in the same manner as above.
Results [0155]
Table 2 shows the results of the test using the combination of the primers of the present invention. This combination of primers does not amplify DNAs other than those of Shiga toxin-producing [0156] Shigella dysenteriae and Verocytotoxin-1-producing Escherishia coli. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with DNAs of the bacteria having the Shiga toxin gene.

The agarose gel electrophoresis used in the above examples of the present invention can differentiate nucleotide fragments from one another which are different in length by 5-10 bases (base pairs) for nucleotide fragments of not more than 100 bases (base pairs), and by 10-20 bases (base pairs) for nucleotide fragments of 100-500 bases (base pairs). In addition, the use of other gel material such as acrylamide makes it possible to improve the precision in measuring the length of nucleotide fragment. Thus, the reliability of the selective detection of the target gene in the present invention can further be increased.

TABLE 2


No	Strains	PCR

01	Bacillus cereus	ATCC14579	−
02	Bacillus Subtills	JCM1465	−
03	Staphylococcus aureus	JCM2413	−
04	Staphylococcus epidermidis	JCM2414	−
05	Salmonella typhimurium	IFO12529	−
06	Salmonella enteritidis	IFO3163	−
07	Clostridium perfringens	ATCC12917	−
08	Vibrio cholerae	ATCC25872	−
09	Vibrio cholerae type Ogawa	ATCC9458	−
10	Vibrio cholerae type Inaba	ATCC9459	−
11	Vibrio fluvialis	JCM3752	−
12	Campylobacter jejuni	JCM2013	−
13	Campylobacter coli	JCM2529	−
14	Escherichia coil	JCM1649	−
15	Yersinia enterocolitica	ATCC9610	−
16	Shigella flexneri	ATCC29903	−
17	Shigella sonnei	ATCC29930	−
18	Bacteroides flagilis	ATCC23745	−
19	Bacteroides vulgatus	JCM5826	−
20	Enterococcus faecalis	JCM5803	−
21	Klebsiella pneumoniae	JCM1662	−
22	Proteus vulgaris	JCM1668	−
23	Citrobacter freundii	ATCC33128	−
24	Streptococcus pyogenes	ATCC12344	−
25	Streptococcus pneumoniae	ATCC33400	−
26	Elaemophilis influenzae	ATCC33391	−
27	Proteus mirabilis	ATCC29906	−
28	Neisseria meningitidis	ATCC13077	−
29	Neisseria gonorrhoeae	ATCC19424	−
30	Listeria monocytogenes	ATCC15313	−
31	Lactobacillus acidophilus	JCM1132	−
32	Bifidobacterium adolescentis	JCM1275	−
33	Fusobacterium nucleatum	ATCC25586	−
34	Propionibacterium acnes	ATCC6919	−
35	Veillonella atypica	ATCC17744	−
36	Pseudomonas aeruginosa	IFO12689	−
37	Corynebacterium diphtheriae	JCM1310	−
38	Peptostreptococcus anaerobius	ATCC27337	−
39	Human placental DNA		−

Example 2

Detection of Shigella Species and EIEC both Having the ipaH Gene

[Experiment 1][0158]
Preparation of Specimens [0159]
The same procedure as used in Example 1 is followed except that 341 strains of Shigella species and EIEC listed in Tables 3-1 to 3-7 are used. [0160]
Synthesis of Primers [0161]
As primers for amplifying the ipaH gene of Shigella species and EIEC strains, the above-described oligonucleotides SEQ ID NO: 3 and SEQ ID NO: 4 are selected based upon the known base sequence of the ipaH gene [Hartman, A. B., et al., J. Bacteriol., 172, 1905-1915(1990); Venkatesan, M. M., et al., Mol. Microbiol., 5, 2435-2446 (1991)]. These oligonucleotides are chemically synthesized by the same method as in [0162] Experiment 1 of Example 1.
PCR [0163]
PCR is carried out under the same reaction conditions as in Example 1 except that the following oligonucleotide combination is used:[0164]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 3+Oligonucleotide SEQ ID NO: 4[0165]
Detection [0166]
Agarose Gel Electrophoresis [0167]
The same procedure as in Example 1 is followed. [0168]
Colony Hybridization Test [0169]
A colony hybridization test is carried out using an oligonucleotide probe specific to the ipaH gene according to the procedure described by Grunstein [Grunstein, M. and Hogness, D., Proc. Natl. Acad. Sci., 72, 3961(1975)]. [0170]
Results [0171]

The base sequence of the ipaH gene of Shigella species and EIEC has already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when the oligonucleotides SEQ ID NO: 3 and SEQ ID NO: 4 of the present invention are used in combination, a nucleotide fragment of 242 bases (or a nucleotide duplex of 242 base pairs) should be amplified. When this estimation accords with the length of the amplified nucleotide fragment, it is judged that PCR using the combination of primers accurately amplify the target region in the ipaH gene, and that the bacterial strain in the specimen has the ipaH gene. The results obtained from the agarose gel electrophoresis with 341 test strains are given in Tables 3-1 to 3-7. PCR using the primers of the present invention amplifies only the DNA of the strains which give the ipaH positive results in the colony hybridization test, showing no amplification of the DNAs of ipaH negative strains. This indicates that PCR using the primers of the present invention is capable of accurately amplifying the ipaH gene and that Shigella species and EIEC both having the ipaH gene can be detected with high accuracy by using the oligonucleotides of the present invention.

TABLE 3-1


No	Strains	CH test*	Primer 3 + 4**

001	S. dysenteriae	TUMD 1	+	+
002	S. dysenteriae	TUMD 2	+	+
003	S. dysenteriae	TUMD 3	+	+
004	S. dysenteriae	TUMD 4	+	+
005	S. dysenteriae	TUMD 5	+	+
006	S. dysenteriae	TUMD 6	+	+
007	S. dysenteriae	MARABIA	−	−
008	S. dysenteriae	AQ-7003	+	+
009	S. dysenteriae	AQ-7004	+	+
010	S. dysenteriae	AQ-7018	+	+
011	S. dysenteriae	AQ-7029	+	+
012	S. dysenteriae	AQ-7030	+	+
013	S. dysenteriae	AQ-7061	+	+
014	S. dysenteriae	AQ-7125	+	+
015	S. dysenteriae	AQ-7131	+	+
016	S. dysenteriae	AQ-7151	+	+
017	S. dysenteriae	AQ-7164	+	+
018	S. dysenteriae	AQ-7166	+	+
019	S. dysenteriae	AQ-7234	+	+
020	S. dysenteriae	AQ-7302	+	+
021	S. dysenteriae	AQ-7350	+	+
022	S. dysenteriae	AQ-7370	+	+
023	S. dysenteriae	AQ-7403	+	+
024	S. dysenteriae	AA-22021	+	+
025	S. dysenteriae	AA-22184	+	+
026	S. dysenteriae	AA-22192	+	−
027	S. dysenteriae	AA-22555	+	−
028	S. dysenteriae	AA-21933	+	+
029	S. dysenteriae	AA-22496	+	+
030	S. dysenteriae	AA-22224	+	+
031	S. dysenteriae	AA-22542	+	+
032	S. dysenteriae	AA-22616	−	+
033	S. dysenteriae	AA-22239	+	+
034	S. dysenteriae	AA-22538	+	+
035	S. dysenteriae	ATCC9361	+	+
036	S. dysenteriae	ATCC9753	+	+
037	S. dysenteriae	ATCC11456a	+	+
038	S. dysenteriae	ATCC13313	+	+
039	S. dysenteriae	ATCC23351	+	+
040	S. dysenteriae	ATCC29027	+	+
041	S. dysenteriae	ATCC29028	+	+
042	S. flexneri	TUMD 7	+	+
043	S. flexneri	TUMD 8	+	+
044	S. flexneri	TUMD 9	+	+
045	S. flexneri	TUMD 10	+	+
046	S. flexneri	TUMD 11	+	+
047	S. flexneri	TUMD 12	+	+
048	S. flexneri	TUMD 13	+	+
049	S. flexneri	TUMD 14	+	+
050	S. flexneri	TUMD 15	+	+

TABLE 3-2


No	Strains	CH test*	Primer 3 + 4**

051	S. flexneri	TUMD 16	+	+
052	S. flexneri	TUMD 17	+	+
053	S. flexneri	TUMD 18	+	+
054	S. flexneri	TUMD 19	+	+
055	S. flexneri	TUMD 20	+	+
056	S. flexneri	TUMD 21	+	+
057	S. flexneri	TUMD 22	+	+
058	S. flexneri	TUMD 23	+	+
059	S. flexneri	TUMD 24	+	+
060	S. flexneri	TUMD 25	+	+
061	S. flexneri	TUMD 26	+	+
062	S. flexneri	TUMD 27	+	+
063	S. flexneri	TUMD 28	+	+
064	S. flexneri	TUMD 29	+	+
065	S. flexneri	TUMD 30	+	+
066	S. flexneri	TUMD 31	+	+
067	S. flexneri	TUMD 32	+	+
068	S. flexneri	TUMD 33	+	+
069	S. flexneri	TUMD 34	+	+
070	S. flexneri	TUMD 35	+	+
071	S. flexneri	TUMD 36	+	+
072	S. flexneri	TUMD 38	+	+
073	S. flexneri	TUMD 39	+	+
074	S. flexneri	TUMD 40	+	+
075	S. flexneri	TUMD 41	+	+
076	S. flexneri	TUMD 42	+	+
077	S. flexneri	TUMD 43	+	+
078	S. flexneri	TUMD 44	+	+
079	S. flexneri	TUMD 45	+	+
080	S. flexneri	TUMD 46	+	+
081	S. flexneri	TUMD 47	+	+
082	S. flexneri	TUMD 48	+	+
083	S. flexneri	TUMD 49	+	+
084	S. flexneri	TUMD 50	+	+
085	S. flexneri	TUMD 51	+	+
086	S. flexneri	TUMD 52	+	+
087	S. flexneri	TUMD 53	+	+
088	S. flexneri	TUMD 54	+	+
089	S. flexneri	TUMD 55	+	+
090	S. flexneri	TUMD 56	+	+
091	S. flexneri	TUMD 57	+	+
092	S. flexneri	TUMD 58	+	+
093	S. flexneri	TUMD 59	+	+
094	S. flexneri	TUMD 60	+	+
095	S. flexneri	TUMD 61	+	+
096	S. flexneri	Maramba 89-77	+	+
097	S. flexneri	Maramba 89-95	+	+
098	S. flexneri	Maramba 89-109a	+	+
099	S. flexneri	Maramba 89-119	+	+
100	S. flexneri	Maramba 89-155	+	+

TABLE 3-3


No	Strains	CH test*	Primer 3 + 4**

101	S. flexneri	Maramba 89-164	+	+
102	S. flexneri	Maramba 89-150	+	+
103	S. flexneri	AA-22175	+	+
104	S. flexneri	AA-22371	+	+
105	S. flexneri	AA-22266	+	+
106	S. flexneri	AA-22636	+	+
107	S. flexneri	AA-22187	+	+
108	S. flexneri	AA-22170	+	+
109	S. flexneri	AA-22367	+	+
110	S. flexneri	AA-22316	+	+
111	S. flexneri	AA-22265	+	+
112	S. flexneri	AA-22296	+	+
113	S. flexneri	AA-22312	+	+
114	S. flexneri	AA-22246	+	+
115	S. flexneri	AA-21981	+	+
116	S. flexneri	AA-22097	+	+
117	S. flexneri	AQ-7347	+	+
118	S. flexneri	AQ-7348	+	+
119	S. flexneri	AQ-7351	+	+
120	S. flexneri	AQ-7360	+	+
121	S. flexneri	AQ-7367	+	+
122	S. flexneri	AQ-7372	+	+
123	S. flexneri	AQ-7378	+	+
124	S. flexneri	AQ-7379	+	+
125	S. flexneri	AQ-7380	+	+
126	S. flexneri	AQ-7385	+	+
127	S. flexneri	AQ-7386	+	+
128	S. flexneri	AQ-7390	+	+
129	S. flexneri	AQ-7391	+	+
130	S. flexneri	AQ-7393	+	+
131	S. flexneri	AQ-7394	+	+
132	S. flexneri	AQ-7398	+	+
133	S. flexneri	AQ-7399	+	+
134	S. flexneri	AQ-7400	+	+
135	S. flexneri	AQ-7402	+	+
136	S. flexneri	AQ-7407	+	+
137	S. flexneri	AQ-7408	+	+
138	S. flexneri	AQ-7411	+	+
139	S. flexneri	AQ-7416	+	+
140	S. flexneri	AQ-7417	+	+
141	S. flexneri	AQ-7418	+	+
142	S. flexneri	AQ-7423	+	+
143	S. flexneri	AQ-7424	+	+
144	S. flexneri	AQ-7426	+	+
145	S. flexneri	AQ-7427	+	+
146	S. flexneri	Manila 89-164	+	−
147	S. flexneri	Manila 89-177	+	+
148	S. flexneri	Manila 89-209	+	+
149	S. flexneri	Manila 89-210	+	+
150	S. flexneri	Manila 89-229	+	+

TABLE 3-4


No	Strains	CH test*	Primer 3 + 4**

151	S. flexneri	Manila 89-230	−	−
152	S. flexneri	Manila 89-231	+	+
153	S. flexneri	Manila 89-232	+	+
154	S. flexneri	Manila 89-233	+	+
155	S. flexneri	Manila 89-273	+	+
156	S. flexneri	Manila 89-328	+	+
157	S. flexneri	Manila 89-333	+	+
158	S. flexneri	Manila 89-365	+	+
159	S. flexneri	Manila 89-274	+	+
160	S. flexneri	Manila 89-436	+	+
161	S. flexneri	Manila 89-438	+	+
162	S. flexneri	Manila 89-443	+	+
163	S. flexneri	Manila 89-444	+	+
164	S. flexneri	Manila 89-450	+	+
165	S. flexneri	Manila 89-480	+	+
166	S. flexneri	Manila 89-483	+	+
167	S. flexneri	Manila 89-486	+	+
168	S. flexneri	Manila 89-498	+	+
169	S. flexneri	Manila 89-499	−	−
170	S. flexneri	Manila 89-503	+	+
171	S. flexneri	Manila 89-509	+	+
172	S. flexneri	Manila 89-532	+	+
173	S. flexneri	Manila 89-539	−	−
174	S. boydii	TUMD 62	+	+
175	S. boydii	TUMD 63	+	+
176	S. boydii	TUMD 64	+	+
177	S. boydii	TUMD 65	+	+
178	S. boydii	TUMD 66	+	+
179	S. boydii	TUMD 67	+	+
180	S. boydii	TUMD 68	+	+
181	S. boydii	AQ-7019	+	+
182	S. boydii	AQ-7020	+	+
183	S. boydii	AQ-7026	+	+
184	S. boydii	AQ-7032	+	+
185	S. boydii	AQ-7039	+	+
186	S. boydii	AQ-7042	+	+
187	S. boydii	AQ-7062	+	+
188	S. boydii	AQ-7076	+	+
189	S. boydii	AQ-7098	+	+
190	S. boydii	AQ-7157	+	+
191	S. boydii	AQ-7193	+	+
192	S. boydii	AQ-7206	+	+
193	S. boydii	AQ-7213	+	+
194	S. boydii	AQ-7218	+	+
195	S. boydii	AQ-7238	+	+
196	S. boydii	AQ-7267	+	+
187	S. boydii	AQ-7268	+	+
198	S. boydii	AQ-7307	+	+
199	S. boydii	AQ-7313	+	+
200	S. boydii	AQ-7314	+	+

TABLE 3-5


No	Strains	CH test*	Primer 3 + 4**

201	S. boydii	AQ-7324	−	−
202	S. boydii	AQ-7349	+	+
203	S. boydii	AQ-7354	+	+
204	S. boydii	AQ-7356	+	+
205	S. boydii	AQ-7357	+	+
206	S. boydii	AQ-7368	+	+
207	S. boydii	AQ-7373	+	+
208	S. boydii	AQ-7376	+	+
209	S. boydii	AQ-7405	+	+
210	S. boydii	AA-22562	+	+
211	S. boydii	AA-22241	−	−
212	S. boydii	AA-22610	−	−
213	S. boydii	AA-20255	+	+
214	S. boydii	AA-20211	+	+
215	S. boydii	AA-21713	+	+
216	S. boydii	AA-17405	−	−
217	S. boydii	AA-22804	+	+
218	S. boydii	AQ-7297	+	+
219	S. sonnei	AQ-7366	+	+
220	S. sonnei	AQ-7369	+	+
221	S. sonnei	AQ-7371	+	+
222	S. sonnei	AQ-7374	+	+
223	S. sonnei	AQ-7375	+	+
224	S. sonnei	AQ-7377	+	+
225	S. sonnei	AQ-7381	+	+
226	S. sonnei	AQ-7382	+	+
227	S. sonnei	AQ-7383	+	+
228	S. sonnei	AQ-7384	+	+
229	S. sonnei	AQ-7387	+	+
230	S. sonnei	AQ-7388	+	+
231	S. sonnei	AQ-7389	+	+
232	S. sonnei	AQ-7392	+	+
233	S. sonnei	AQ-7395	+	+
234	S. sonnei	AQ-7396	+	+
235	S. sonnei	AQ-7397	+	+
236	S. sonnei	AQ-7401	+	+
237	S. sonnei	AQ-7406	+	+
238	S. sonnei	AQ-7409	+	+
239	S. sonnei	AQ-7410	+	+
240	S. sonnei	AQ-7412	+	+
241	S. sonnei	AQ-7413	+	+
242	S. sonnei	AQ-7414	+	+
243	S. sonnei	AQ-7415	+	+
244	S. sonnei	AQ-7419	+	+
245	S. sonnei	AQ-7420	+	+
246	S. sonnei	AQ-7421	+	+
247	S. sonnei	AQ-7422	+	+
248	S. sonnei	AQ-7425	+	+
249	S. sonnei	AA-22634	+	+
250	S. sonnei	AA-22677	+	+

TABLE 3-6


No	Strains	CH test*	Primer 3 + 4**

251	S. sonnei	AA-18306	+	+
252	S. sonnei	AA-22067	+	+
253	S. sonnei	AA-22870	+	+
254	S. sonnei	TUMD 69	+	+
255	S. sonnei	TUMD 70	+	+
256	S. sonnei	TUMD 71	+	+
257	S. sonnei	TUMD 72	+	+
258	S. sonnei	TUMD 73	+	+
259	S. sonnei	TUMD 74	+	+
260	S. sonnei	TUMD 75	+	+
261	S. sonnei	TUMD 76	+	+
262	S. sonnei	TUMD 77	+	+
263	S. sonnei	TUMD 78	+	+
264	S. sonnei	TUMD 79	+	+
265	S. sonnei	TUMD 80	+	+
266	S. sonnei	TUMD 81	+	+
267	S. sonnei	TUMD 82	+	+
268	S. sonnei	TUMD 83	+	+
269	S. sonnei	TUMD 84	+	+
270	S. sonnei	TUMD 85	+	+
271	S. sonnei	TUMD 86	+	+
272	S. sonnei	TUMD 87	+	+
273	S. sonnei	TUMD 88	+	+
274	S. sonnei	TUMD 89	+	+
275	S. sonnei	TUMD 90	+	+
276	S. sonnei	TUMD 91	+	+
277	S. sonnei	TUMD 92	+	−
228	S. sonnei	TUMD 93	+	+
279	S. sonnei	TUMD 94	+	+
280	S. sonnei	TUMD 95	+	+
281	S. sonnei	TUMD 96	+	+
282	S. sonnei	TUMD 97	+	+
283	S. sonnei	TUMD 98	+	+
284	S. sonnei	TUMD 99	+	+
285	S. sonnei	TUMD 100	+	+
286	S. sonnei	TUMD 101	+	+
287	S. sonnei	TUMD 102	+	+
288	S. sonnei	TUMD 103	+	+
289	S. sonnei	TUMD 104	+	+
290	S. sonnei	TUMD 105	+	+
291	S. sonnei	TUMD 106	+	+
292	S. sonnei	TUMD 107	+	+
293	S. sonnei	TUMD 108	+	+
294	S. sonnei	TUMD 109	+	+
295	S. sonnei	TUMD 110	+	+
296	S. sonnei	TUMD 111	+	+
297	S. sonnei	TUMD 112	+	+
298	S. sonnei	TUMD 113	+	+
299	S. sonnei	TUMD 114	+	+
200	S. sonnei	TUMD 115	+	+

TABLE 3-7


No	Strains	CH test*	Primer 3 + 4**

301	S. sonnei	TUMD 116	+	+
302	S. sonnei	TUMD 117	+	+
303	S. sonnei	TUMD 118	+	+
304	S. sonnei	TUMD 119	+	+
305	S. sonnei	TUMD 120	+	+
306	S. sonnei	TUMD 121	+	+
307	S. sonnei	TUMD 122	+	+
308	S. sonnei	TUMD 123	+	+
309	S. sonnei	TUMD 124	+	+
310	S. sonnei	TUMD 125	+	+
311	S. sonnei	TUMD 126	+	+
312	S. sonnei	TUMD 127	+	+
313	S. sonnei	TUMD 128	+	+
314	S. sonnei	Maramba 89-154	+	+
315	S. sonnei	Maramba 89-161	+	+
316	S. sonnei	Manila 89-342	+	+
317	S. sonnei	Manila 89-445	+	+
318	E. coli	DMR 6	+	+
319	E. coli	DMR 78	+	+
320	E. coli	DMR 79	+	+
321	E. coli	AQ8001	+	+
322	E. coli	AQ8003	+	+
323	E. coli	AQ8004	+	+
324	E. coli	AQ8008	+	+
325	E. coli	AQ8010	+	+
326	E. coli	AQ8011	+	+
327	E. coli	AQ8012	+	+
328	E. coli	AQ8013	+	+
329	E. coli	AQ8016	+	+
330	E. coli	AQ8019	+	+
331	E. coli	AQ8022	+	+
332	E. coli	AQ8024	+	+
333	E. coli	AQ8025	+	+
334	E. coli	AQ8027	+	+
335	E. coli	AQ8028	+	+
336	E. coli	AQ8029	+	+
337	E. coli	AQ8031	+	+
338	E. coli	AQ8033	−	−
339	E. coli	AQ8036	−	−
340	E. coli	AQ8044	+	+
341	E. coli	PE660	+	+

[Experiment 2][0179]
To determine whether the results obtained in [0180] Experiment 1 are specific to the ipaH gene, the DNAs of clinically important pathogenic bacteria other than Shigella species and EIEC are examined with the primers of the present invention. The same procedure as used in Experiment 1 is followed, except for the procedure of preparation of specimens.
Preparation of Specimens [0181]
Each strain listed in Table 4 is treated in the same manner as in [0182] Experiment 2 of Example 1.
Results [0183]

Table 4 shows the results of the test using the combination of primers of the present invention. This combination of primers does not amplify any DNAs other than those of Shigella species and EIEC. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with DNAs of the bacteria having the ipaH gene.

TABLE 4


No	Strains	PCR

01	Bacillus cereus	ATCC14579	−
02	Bacillus Subtillis	JCM1465	−
03	Staphylococcus aureus	JCM2413	−
04	Staphylococcus epidermidis	JCM2414	−
05	Salmonella typhimurium	IFO12529	−
06	Salmonella enteritidis	IFO3163	−
07	Clostridium perfringens	ATCC12917	−
08	Vibrio cholerae	ATCC25872	−
09	Vibrio cholerae type Ogawa	ATCC9458	−
10	Vibrio cholerae type Inaba	ATCC9459	−
11	Vibrio fluvialis	JCM3752	−
12	Campylobacter jejuni	JCM2013	−
13	Campylobacter coli	JCM2529	−
14	Escherichia coli	JCM1649	−
15	Yersinia enterocolitica	ATCC9610	−
16	Corynebacterium diphtheriae	JCM13	−
17	Peptostreptococcus anaerobius	ATCC23745	−
18	Bacteroides flagilis	ATCC23745	−
19	Bacteroides vulgatus	JCM5826	−
20	Enterococcus faecalis	JCM5803	−
21	Klebsiella pneumoniae	JCM1662	−
22	Proteus vulgaris	JCM1668	−
23	Citrobacter freundii	ATCC33128	−
24	Streptococcus pyogenes	ATCC12344	−
25	Streptococcus pneumoniae	ATCC33400	−
26	Elaemophilis influenzae	ATCC33391	−
27	Proteus mirabilis	ATCC29906	−
28	Neisseria meningitidis	ATCC13077	−
29	Neisseria gonorrhoeae	ATCC19424	−
30	Listeria monocytogenes	ATCC15313	−
31	Lactobacillus acidophilus	JCM1132	−
32	Bifidobacterium adolescentis	JCM1275	−
33	Fusobacterium nucleatum	ATCC25586	−
34	Propionibacterium acnes	ATCC6919	−
35	Veillonella atypica	ATCC17744	−
36	Pseudomonas aeruginosa	IFO12689	−
37	Human placental DNA		−

Example 3

Detection of Shigella Species and EIEC Having the invE Gene

[Experiment 1][0185]
Preparation of Specimens [0186]
The same procedure as used in Example 1 is followed except that 341 strains of Shigella species and EIEC listed in Tables 3-1 to 3-7 are used. [0187]
Synthesis of Primers [0188]
As primers for amplifying the invE gene of Shigella species and EIEC, the above-described oligonucleotides SEQ ID NO: 5 and SEQ ID NO: 6 are selected based upon the known base sequence of the invE gene [Watanabe, H., et al., J. Bacteriol., 172, 619-629(1990)]. These oligonucleotides are chemically synthesized by the same method as in [0189] Experiment 1 of Example 1.
PCR [0190]
PCR is carried out under the same reaction conditions as in Example 1 except that the following oligonucleotide combination is used: [0191]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 5+Oligonucleotide SEQ ID NO: 6[0192]
Detection [0193]
Agarose Gel Electrophoresis [0194]
The same procedure as in Example 1 is followed. [0195]
Colony Hybridization Test [0196]
A colony hybridization test is carried out using an oligonucleotide probe specific to invE gene according to the procedure described by Grunstein [Grunstein, M. and Hogness, D., Proc. Natl. Acad. Sci., 72, 3961(1975)]. [0197]
Results [0198]

The base sequence of the invE gene of Shigella species and EIEC has already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, the oligonucleotides SEQ ID NO: 5 and SEQ ID NO: 6 of the present invention are used in combination, a nucleotide fragment of 293 bases (or a nucleotide duplex of 293 base pairs) should be amplified. When this estimation accords with the length of the amplified nucleotide sequence, it is judged that the combination of primers accurately amplifies the target region in the invE gene, and that the bacterial strain in the specimen has the invE gene. The results obtained from the agarose gel electrophoresis with 341 test strains are given in Tables 5-1 to 5-7. PCR using the primers of the present invention amplifies only the DNA of the strains which give the invE positive results in the colony hybridization test, showing no amplification of DNAs of invE negative strains. This indicates that PCR using the primers of the present invention is capable of accurately amplifying the invE gene and that Shigella species and EIEC both having the invE gene can be detected with high accuracy by using the oligonucleotides of the present invention.

TABLE 5 - 1


		CH	Primer
No	Strains	test*	5 + 6**

001	S. dysenteriae	TUMD 1	−	−
002	S. dysenteriae	TUMD 2	−	−
003	S. dysenteriae	TUMD 3	−	−
004	S. dysenteriae	TUMD 4	+	+
005	S. dysenteriae	TUMD 5	−	−
006	S. dysenteriae	TUMD 6	−	−
007	S. dysenteriae	MARABIA	−	−
008	S. dysenteriae	AQ-7003	−	−
009	S. dysenteriae	AQ-7004	+	+
010	S. dysenteriae	AQ-7018	−	−
011	S. dysenteriae	AQ-7029	−	−
012	S. dysenteriae	AQ-7030	+	+
013	S. dysenteriae	AQ-7061	−	−
014	S. dysenteriae	AQ-7125	+	+
015	S. dysenteriae	AQ-7131	−	−
016	S. dysenteriae	AQ-7151	−	−
017	S. dysenteriae	AQ-7164	+	+
018	S. dysenteriae	AQ-7166	+	+
019	S. dysenteriae	AQ-7234	+	+
020	S. dysenteriae	AQ-7302	−	−
021	S. dysenteriae	AQ-7350	+	+
022	S. dysenteriae	AQ-7370	+	+
023	S. dysenteriae	AQ-7403	+	+
024	S. dysenteriae	AA-22021	+	+
025	S. dysenteriae	AA-22184	−	−
026	S. dysenteriae	AA-22192	−	−
027	S. dysenteriae	AA-22555	+	+
028	S. dysenteriae	AA-21933	+	+
029	S. dysenteriae	AA-22496	+	+
030	S. dysenteriae	AA-22224	−	−
031	S. dysenteriae	AA-22542	+	+
032	S. dysenteriae	AA-22616	−	−
033	S. dysenteriae	AA-22239	+	+
034	S. dysenteriae	AA-22538	+	+
035	S. dysenteriae	ATCC9361	+	+
036	S. dysenteriae	ATCC9753	+	+
037	S. dysenteriae	ATCC11456a	+	+
038	S. dysenteriae	ATCC13313	+	+
039	S. dysenteriae	ATCC23351	+	+
040	S. dysenteriae	ATCC29027	+	+
041	S. dysenteriae	ATCC29028	+	+
042	S. flexneri	TUMD 7	−	−
043	S. flexneri	TUMD 8	−	−
044	S. flexneri	TUMD 9	+	+
045	S. flexneri	TUMD10	−	−
046	S. flexneri	TUMD11	−	−
047	S. flexneri	TUMD12	+	+
048	S. flexneri	TUMD13	+	+
049	S. flexneri	TUMD14	−	−
050	S. flexneri	TUMD15	+	+

TABLE 5 - 2


		CH	Primer
No	Strains	test*	5 + 6**

051	S. flexneri	TUMD16	−	−
052	S. flexneri	TUMD17	−	−
053	S. flexneri	TUMD18	−	−
054	S. flexneri	TUMD19	+	+
055	S. flexneri	TUMD20	−	−
056	S. flexneri	TUMD21	−	−
057	S. flexneri	TUMD22	−	−
058	S. flexneri	TUMD23	−	−
059	S. flexneri	TUMD24	+	+
060	S. flexneri	TUMD25	−	−
061	S. flexneri	TUMD26	−	−
062	S. flexneri	TUMD27	−	−
063	S. flexneri	TUMD28	−	−
064	S. flexneri	TUMD29	−	−
065	S. flexneri	TUMD30	+	+
066	S. flexneri	TUMD31	−	−
067	S. flexneri	TUMD32	−	−
068	S. flexneri	TUMD33	−	−
069	S. flexneri	TUMD34	−	−
070	S. flexneri	TUMD35	+	+
071	S. flexneri	TUMD36	+	+
072	S. flexneri	TUMD38	−	−
073	S. flexneri	TUMD39	+	+
074	S. flexneri	TUMD40	−	−
075	S. flexneri	TUMD41	+	+
076	S. flexneri	TUMD42	+	+
077	S. flexneri	TUMD43	+	+
078	S. flexneri	TUMD44	+	+
079	S. flexneri	TUMD45	+	+
080	S. flexneri	TUMD46	−	−
081	S. flexneri	TUMD47	+	+
082	S. flexneri	TUMD48	−	−
083	S. flexneri	TUMD49	+	+
084	S. flexneri	TUMD50	−	−
085	S. flexneri	TUMD51	−	−
086	S. flexneri	TUMD52	+	+
087	S. flexneri	TUMD53	−	−
088	S. flexneri	TUMD54	−	−
089	S. flexneri	TUMD55	−	−
090	S. flexneri	TUMD56	−	−
091	S. flexneri	TUMD57	−	−
092	S. flexneri	TUMD58	−	−
093	S. flexneri	TUMD59	−	−
094	S. flexneri	TUMD60	−	−
095	S. flexneri	TUMD61	−	−
096	S. flexneri	Maramba 89-77	−	−
097	S. flexneri	Maramba 89-95	+	+
098	S. flexneri	Maramba 89-109a	−	−
099	S. flexneri	Maramba 89-119	−	−
100	S. flexneri	Maramba 89-155	−	−

TABLE 5 - 3


		CH	Primer
No	Strains	test*	5 + 6**

101	S. flexneri	Maramba 89-164	−	−
102	S. flexneri	Maramba 89-150	+	+
103	S. flexneri	AA-22175	−	−
104	S. flexneri	AA-22371	−	−
105	S. flexneri	AA-22266	+	+
106	S. flexneri	AA-22636	+	+
107	S. flexneri	AA-22187	−	−
108	S. flexneri	AA-22170	+	+
109	S. flexneri	AA-22367	+	+
110	S. flexneri	AA-22316	−	−
111	S. flexneri	AA-22265	+	+
112	S. flexneri	AA-22296	−	−
113	S. flexneri	AA-22312	+	+
114	S. flexneri	AA-22246	−	−
115	S. flexneri	AA-21981	−	−
116	S. flexneri	AA-22097	−	−
117	S. flexneri	AQ-7347	+	+
118	S. flexneri	AQ-7348	+	+
119	S. flexneri	AQ-7351	−	−
120	S. flexneri	AQ-7360	+	+
121	S. flexneri	AQ-7367	+	+
122	S. flexneri	AQ-7372	+	+
123	S. flexneri	AQ-7378	−	−
124	S. flexneri	AQ-7379	−	−
125	S. flexneri	AQ-7380	−	−
126	S. flexneri	AQ-7385	+	+
127	S. flexneri	AQ-7386	−	−
128	S. flexneri	AQ-7390	−	−
129	S. flexneri	AQ-7391	+	+
130	S. flexneri	AQ-7393	−	−
131	S. flexneri	AQ-7394	−	−
132	S. flexneri	AQ-7398	−	−
133	S. flexneri	AQ-7399	−	−
134	S. flexneri	AQ-7400	−	−
135	S. flexneri	AQ-7402	−	−
136	S. flexneri	AQ-7407	−	−
137	S. flexneri	AQ-7408	−	−
138	S. flexneri	AQ-7411	+	+
139	S. flexneri	AQ-7416	+	+
140	S. flexneri	AQ-7417	+	+
141	S. flexneri	AQ-7418	+	+
142	S. flexneri	AQ-7423	+	+
143	S. flexneri	AQ-7424	−	−
144	S. flexneri	AQ-7426	+	+
145	S. flexneri	AQ-7427	+	+
146	S. flexneri	Manila 89-164	−	−
147	S. flexneri	Manila 89-177	−	−
148	S. flexneri	Manila 89-209	−	−
149	S. flexneri	Manila 89-210	+	+
150	S. flexneri	Manila 89-229	+	+

TABLE 5 - 4


		CH	Primer
No	Strains	test*	5 + 6**

151	S. flexneri	Manila 89-230	−	−
152	S. flexneri	Manila 89-231	+	+
153	S. flexneri	Manila 89-232	+	+
154	S. flexneri	Manila 89-233	−	−
155	S. flexneri	Manila 89-273	+	+
156	S. flexneri	Manila 89-328	+	+
157	S. flexneri	Manila 89-333	+	+
158	S. flexneri	Manila 89-365	+	+
159	S. flexneri	Manila 89-274	−	−
160	S. flexneri	Manila 89-436	−	−
161	S. flexneri	Manila 89-438	+	+
162	S. flexneri	Manila 89-443	+	+
163	S. flexneri	Manila 89-444	+	+
164	S. flexneri	Manila 89-450	+	+
165	S. flexneri	Manila 89-480	+	+
166	S. flexneri	Manila 89-483	−	−
167	S. flexneri	Manila 89-486	−	−
168	S. flexneri	Manila 89-498	−	−
169	S. flexneri	Manila 89-499	−	−
170	S. flexneri	Manila 89-503	−	−
171	S. flexneri	Manila 89-509	−	−
172	S. flexneri	Manila 89-532	−	−
173	S. flexneri	Manila 89-539	−	−
174	S. boydii	TUMD62	−	−
175	S. boydii	TUMD63	+	+
176	S. boydii	TUMD64	+	+
177	S. boydii	TUMD65	−	−
178	S. boydii	TUMD66	+	+
179	S. boydii	TUMD67	−	−
180	S. boydii	TUMD68	−	−
181	S. boydii	AQ-7019	−	−
182	S. boydii	AQ-7020	−	−
183	S. boydii	AQ-7026	−	−
184	S. boydii	AQ-7032	+	+
185	S. boydii	AQ-7039	+	+
186	S. boydii	AQ-7042	+	+
187	S. boydii	AQ-7062	+	+
188	S. boydii	AQ-7076	−	−
189	S. boydii	AQ-7098	+	+
190	S. boydii	AQ-7157	+	+
191	S. boydii	AQ-7193	−	−
192	S. boydii	AQ-7206	−	−
193	S. boydii	AQ-7213	+	+
194	S. boydii	AQ-7218	+	+
195	S. boydii	AQ-7238	+	+
196	S. boydii	AQ-7267	−	−
197	S. boydii	AQ-7268	+	+
198	S. boydii	AQ-7307	+	+
199	S. boydii	AQ-7313	−	−
200	S. boydii	AQ-7314	−	−

TABLE 5 - 5


		CH	Primer
No	Strains	test*	5 + 6**

201	S. boydii	AQ-7324	−	−
202	S. boydii	AQ-7349	+	+
203	S. boydii	AQ-7354	−	−
204	S. boydii	AQ-7356	−	−
205	S. boydii	AQ-7357	+	+
206	S. boydii	AQ-7368	+	+
207	S. boydii	AQ-7373	+	+
208	S. boydii	AQ-7376	−	−
209	S. boydii	AQ-7405	+	+
210	S. boydii	AA-22562	+	+
211	S. boydii	AA-22241	−	−
212	S. boydii	AA-22610	−	−
213	S. boydii	AA-20255	+	+
214	S. boydii	AA-20211	+	+
215	S. boydii	AA-21713	−	−
216	S. boydii	AA-17405	−	−
217	S. boydii	AA-22804	−	−
218	S. boydii	AQ-7297	+	+
219	S. sonnei	AQ-7366	−	−
220	S. sonnei	AQ-7369	+	+
221	S. sonnei	AQ-7371	−	−
222	S. sonnei	AQ-7374	−	−
223	S. sonnei	AQ-7375	+	+
224	S. sonnei	AQ-7377	+	+
225	S. sonnei	AQ-7381	−	−
226	S. sonnei	AQ-7382	−	−
227	S. sonnei	AQ-7383	−	−
228	S. sonnei	AQ-7384	−	−
229	S. sonnei	AQ-7387	−	−
230	S. sonnei	AQ-7388	+	+
231	S. sonnei	AQ-7389	+	+
232	S. sonnei	AQ-7392	+	+
233	S. sonnei	AQ-7395	−	−
234	S. sonnei	AQ-7396	−	−
235	S. sonnei	AQ-7397	+	+
236	S. sonnei	AQ-7401	+	+
237	S. sonnei	AQ-7406	+	+
238	S. sonnei	AQ-7409	−	−
239	S. sonnei	AQ-7410	−	−
240	S. sonnei	AQ-7412	−	−
241	S. sonnei	AQ-7413	+	+
242	S. sonnei	AQ-7414	−	−
243	S. sonnei	AQ-7415	−	−
244	S. sonnei	AQ-7419	+	+
245	S. sonnei	AQ-7420	−	−
246	S. sonnei	AQ-7421	+	+
247	S. sonnei	AQ-7422	+	+
248	S. sonnei	AQ-7425	−	−
249	S. sonnei	AA-22634	+	+
250	S. sonnei	AA-22677	+	+

TABLE 5 - 6


		CH	Primer
No	Strains	test*	5 + 6**

251	S. sonnei	AA-18306	−	−
252	S. sonnei	AA-22067	−	−
253	S. sonnei	AA-22870	−	−
254	S. sonnei	TUMD69	+	+
255	S. sonnei	TUMD70	−	−
256	S. sonnei	TUMD71	−	−
257	S. sonnei	TUMD72	−	−
258	S. sonnei	TUMD73	+	+
259	S. sonnei	TUMD74	−	−
260	S. sonnei	TUMD75	+	+
261	S. sonnei	TUMD76	−	−
262	S. sonnei	TUMD77	−	−
263	S. sonnei	TUMD78	+	+
264	S. sonnei	TUMD79	−	−
265	S. sonnei	TUMD80	−	−
266	S. sonnei	TUMD81	+	+
267	S. sonnei	TUMD82	−	−
268	S. sonnei	TUMD83	−	−
269	S. sonnei	TUMD84	−	−
270	S. sonnei	TUMD85	−	−
271	S. sonnei	TUMD86	−	−
272	S. sonnei	TUMD87	−	−
273	S. sonnei	TUMD88	−	−
274	S. sonnei	TUMD89	−	−
275	S. sonnei	TUMD90	+	+
276	S. sonnei	TUMD91	−	−
277	S. sonnei	TUMD92	+	+
278	S. sonnei	TUMD93	+	+
279	S. sonnei	TUMD94	+	+
280	S. sonnei	TUMD95	−	−
281	S. sonnei	TUMD96	−	−
282	S. sonnei	TUMD97	−	−
283	S. sonnei	TUMD98	−	−
284	S. sonnei	TUMD99	−	−
285	S. sonnei	TUMD100	−	−
286	S. sonnei	TUMD101	−	−
287	S. sonnei	TUMD102	+	+
288	S. sonnei	TUMD103	+	+
289	S. sonnei	TUMD104	+	+
290	S. sonnei	TUMD105	+	+
291	S. sonnei	TUMD106	−	−
292	S. sonnei	TUMD107	+	+
293	S. sonnei	TUMD108	−	−
294	S. sonnei	TUMD109	+	+
295	S. sonnei	TUMD110	−	−
296	S. sonnei	TUMD111	+	+
297	S. sonnei	TUMD112	−	−
298	S. sonnei	TUMD113	+	+
299	S. sonnei	TUMD114	+	+
200	S. sonnei	TUMD115	−	−

TABLE 5 - 7


		CH	Primer
No	Strains	test*	5 + 6**

301	S. sonnei	TUMD116	−	−
302	S. sonnei	TUMD117	−	−
303	S. sonnei	TUMD118	+	+
304	S. sonnei	TUMD119	+	+
305	S. sonnei	TUMD120	+	+
306	S. sonnei	TUMD121	−	−
307	S. sonnei	TUMD122	−	−
308	S. sonnei	TUMD123	+	+
309	S. sonnei	TUMD124	+	+
310	S. sonnei	TUMD125	−	−
311	S. sonnei	TUMD126	+	+
312	S. sonnei	TUMD127	−	−
313	S. sonnei	TUMD128	−	−
314	S. sonnei	Maramba 89-154	+	+
315	S. sonnei	Maramba 89-161	−	−
316	S. sonnei	Manila 89-342	−	−
317	S. sonnei	Manila 89-445	+	+
318	E. coli	DMR 6	+	+
319	E. coli	DMR78	+	+
320	E. coli	DMR79	−	−
321	E. coli	AQ8001	−	−
322	E. coli	AQ8003	+	+
323	E. coli	AQ8004	−	−
324	E. coli	AQ8008	+	+
325	E. coli	AQ8010	+	+
326	E. coli	AQ8011	+	+
327	E. coli	AQ8012	+	+
328	E. coli	AQ8013	−	−
329	E. coli	AQ8016	+	+
330	E. coli	AQ8019	+	+
331	E. coli	AQ8022	+	+
332	E. coli	AQ8024	−	−
333	E. coli	AQ8025	+	+
334	E. coli	AQ8027	−	−
335	E. coli	AQ8028	+	+
336	E. coli	AQ8029	+	+
337	E. coli	AQ8031	+	+
338	E. coli	AQ8033	−	−
339	E. coli	AQ8036	−	−
340	E. coli	AQ8044	+	+
341	E. coli	PE660	+	+

[Experiment 2][0206]
To determine whether the results obtained in [0207] Experiment 1 are specific to the invE gene, DNAs of clinically important pathogenic bacteria other than Shigella species and EIEC are examined with the primers of the present invention. The same procedure as used in Experiment 1 is followed, except for the procedure of preparation of specimens.
Preparation of Specimens [0208]
Each strain listed in Table 6 is treated in the same manner as in [0209] Experiment 2 of Example 1.
Results [0210]

Table 6 shows the results of the test using the combination of primers of the present invention. This combination of primers does not amplify any DNAs of pathogenic bacteria other than Shigella species and EIEC. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with DNAs of the bacteria having the invE gene.

TABLE 6


No	Strains	PCR

01	Bacillus cereus	ATCC14579	—
02	Bacillus Subtilis	JCM1465	—
03	Staphylococcus aureus	JCM2413	—
04	Staphylococcus epidermidis	JCM2414	—
05	Salmonella typhimurium	IF012529	—
06	Salmonella enteritidis	IF03163	—
07	Clostridium perfringens	ATCC12917	—
08	Vibrio cholerae	ATCC25872	—
09	Vibrio cholerae type Ogawa	ATCC9458	—
10	Vibrio cholerae type Inaba	ATCC9459	—
11	Vibrio fluvialis	JCM3752	—
12	Campylobacter jejuni	JCM2013	—
13	Campylobacter coli	JCM2529	—
14	Escherichia coli	JCM1649	—
15	Yersinia enterocolitica	ATCC9610	—
16	Corynebacterium diphtheriae	JCM13	—
17	Peptostreptococcus anaerobius	ATCC273	—
18	Bacteroides flagilis	ATCC23745	—
19	Bacteroides vulgatus	JCM5826	—
20	Enterococcus faecalis	JCM5803	—
21	Klebsiella pneumoniae	JCM1662	—
22	Proteus vulgaris	JCM1668	—
23	Citrobacter freundii	ATCC33128	—
24	Streptococcus pyogenes	ATCC12344	—
25	Streptococcus pneumoniae	ATCC33400	—
26	Elaemophilis influenzae	ATCC33391	—
27	Proteus mirabilis	ATCC29906	—
28	Neisseria meningitidis	ATCC13077	—
29	Neisseria gonorthoeae	ATCC19424	—
30	Listeria monocytogenes	ATCC15313	—
31	Lactobacillus acidophilus	JCM1132	—
32	Bifidobacterium adolescentis	JCM1275	—
33	Fusobacterium nucleatum	ATCC25586	—
34	Propionibacterium acnes	ATCC6919	—
35	Veillonella atypica	ATCC17744	—
36	Pseudomonas aeruginosa	IF012689	—
37	Human placental DNA		—

Example 4

Detection of Salmonella Species having the araC Gene

[Experiment 1][0212]
Preparation of Specimens [0213]
As listed in Tables 7-1 to 7-6, the 133 various Salmonella species isolated from the patients and food samples are used. The details are as follows: 67 strains of [0214] Salmonella typhimurium, 1 of Salmonella havana, 2 of Salmonella oranienburg, 3 of Salmonella london, 3 of Salmonella senftenberg, 4 of Salmonella blockley, 3 of Salmonella agona, 4 of Salmonella infantis, 14 of Salmonella litchfield, 6 of Salmonella enteritidis, 13 of Salmonella thompson, 6 of Samonella paratyphi B, 2 of Salmonella Montevideo, 1 of Salmonella gallinarum, 1 of Salmonella choleraesuis, 1 of Salmonella derby, 1 of Salmonella give and 1 of Salmonella Heidelberg. Each strain is inoculated to an appropriate medium, and subjected to overnight culture at 37° C. under aerobic conditions. Each culture broth is diluted with TE buffer, and heated at 95° C. for 10 minutes, followed by centrifugation. The supernatants are used as specimens.
Synthesis of Primers [0215]
As primers for amplifying the araC gene of [0216] Salmonella typhimurium, the above-described oligonucleotides SEQ ID NO: 7 to SEQ ID NO: 1l are selected based upon the known base sequence of the arac gene [Horwitz, A. H., et al., Gene 14, 309-319(1981); Clarke, P., et al., Gene 18, 157-163(1982); Lee, J. -H., et al., Gene 46, 113-121 (1986)], and chemically synthesized by the same method as in Experiment 1 of Example 1.
PCR [0217]
PCR is carried out under the same reaction conditions as in Example 1 except that any one of the following oligonucleotide combinations is used:[0218]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 7+Oligonucleotide SEQ ID NO: 8; [0219]
Oligonucleotide SEQ ID NO: 9+Oligonucleotide SEQ ID NO: 10; and [0220]
Oligonucleotide SEQ ID NO: 11+Oligonucleotide SEQ ID NO: 8. [0221]
Detection [0222]
Agarose Gel Electrophoresis [0223]
The same procedure as in Example 1 is followed. [0224]
Results [0225]

The base sequence of the araC gene of Salmonella typhimurium has already been determined. This base sequence is thought to be common to all Salmonella species. The length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when oligonucleotides SEQ ID NO: 7 and SEQ ID NO: 8 of the present invention are used in combination, a nucleotide fragment of 361 bases (or a nucleotide duplex of 361 base pairs) is amplified. Similarly, the combination of SEQ ID NO: 9 and SEQ ID NO: 10, and that of SEQ ID NO: 11 and SEQ ID NO: 8 amplify nucleotide fragment of 493 bases and that of 334 bases, respectively. When these estimations accord with the length of the amplified nucleotide fragments, it is judged that PCR using the combination of primers accurately amplifies the target region in the araC gene, and that the bacterial strain in the specimen has the arac gene. Tables 7-1 to 7-6 shows the results of the detection of the arac gene in Salmonella species. As obvious from Tables 7-1 to 7-6, the arac gene of Salmonella species are detected with high accuracy by using the oligonucleotide primers of the present invention.

	TABLE 7 - 1


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

001	Salmonella typhimurium	56-1	+	+	+
002	Salmonella typhimurium	56-2	+	+	+
003	Salmonella typhimurium	56-3	+	+	+
004	Salmonella typhimurium	56-4	+	+	+
005	Salmonella typhimurium	56-5	+	+	+
006	Salmonella typhimurium	56-6	+	+	+
007	Salmonella typhimurium	56-7	+	+	+
008	Salmonella typhimurium	56-11	+	+	+
009	Salmonella typhimurium	56-12	+	+	+
010	Salmonella typhimurium	56-13	+	+	+
011	Salmonella typhimurium	56-17	+	+	+
012	Salmonella typhimurium	56-18	+	+	+
013	Salmonella typhimurium	56-19	+	+	+
014	Salmonella typhimurium	56-20	+	+	+
015	Salmonella typhimurium	56-21	+	+	+
016	Salmonella typhimurium	56-22	+	+	+
017	Salmonella typhimurium	56-23	+	+	+
018	Salmonella typhimurium	56-25	+	+	+
019	Salmonella typhimurium	56-26	+	+	+
020	Salmonella typhimurium	56-27	+	+	+
021	Salmonella typhimurium	56-30	+	+	+
022	Salmonella typhimurium	56-31	+	+	+
023	Salmonella typhimurium	56-32	+	+	+
024	Salmonella typhimurium	57-3	+	+	+
025	Salmonella typhimurium	57-4	+	+	+

	TABLE 7 - 2


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

026	Salmonella typhimurium	57-5	+	+	+
027	Salmonella typhimurium	57-6	+	+	+
028	Salmonella typhimurium	57-7	+	+	+
029	Salmonella typhimurium	57-9	+	+	+
030	Salmonella typhimurium	57-10	+	+	+
031	Salmonella typhimurium	57-11	+	+	+
032	Salmonella typhimurium	57-19	+	+	+
033	Salmonella typhimurium	57-20	+	+	+
034	Salmonella typhimurium	59-26	+	+	+
035	Salmonella typhimurium	59-27	+	+	+
036	Salmonella typhimurium	59-28	+	+	+
037	Salmonella typhimurium	59-54	+	+	+
038	Salmonella typhimurium	59-55	+	+	+
039	Salmonella typhimurium	59-56	+	+	+
040	Salmonella typhimurium	59-57	+	+	+
041	Salmonella typhimurium	59-58	+	+	+
042	Salmonella typhimurium	60-5	+	+	+
043	Salmonella typhimurium	60-6	+	+	+
044	Salmonella typhimurium	60-7	+	+	+
045	Salmonella typhimurium	60-13	+	+	+
046	Salmonella typhimurium	61-1	+	+	+
047	Salmonella typhimurium	61-16	+	+	+
048	Salmonella typhimurium	62-1	+	+	+
049	Salmonella typhimurium	62-2	+	+	+
050	Salmonella typhimurium	62-3	+	+	+

	TABLE 7 - 3


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

051	Salmonella	62-4	+	+	+
	typhimurium
052	Salmonella	62-5	+	+	+
	typhimurium
053	Salmonella	62-6	+	+	+
	typhimurium
054	Salmonella	63-6	+	+	+
	typhimurium
055	Salmonella	63-7	+	+	+
	typhimurium
056	Salmonella	63-8	+	+	+
	typhimurium
057	Salmonella	63-9	+	+	+
	typhimurium
058	Salmonella	89-1	+	+	+
	typhimurium
059	Salmonella	89-2	+	+	+
	typhimurium
060	Salmonella	IFO12529	+	+	+
	typhimurium
061	Salmonella	IFO13245	+	+	+
	typhimurium
062	Salmonella	IFO14193	+	+	+
	typhimurium
063	Salmonella	IFO14194	+	+	+
	typhimurium
064	Salmonella	IFO14209	+	+	+
	typhimurium
065	Salmonella	IFO14210	+	+	+
	typhimurium
066	Salmonella	IFO14211	+	+	+
	typhimurium
067	Salmonella	IFO14212	+	+	+
	typhimurium
068	Salmonella litchfield	56-8	+	+	+
069	Salmonella litchfield	59-25	+	+	+
070	Salmonella litchfield	53-22	+	+	+
071	Salmonella litchfield	53-23	+	+	+
072	Salmonella litchfield	53-24	+	+	+
073	Salmonella litchfield	54-5	+	+	+
074	Salmonella litchfield	54-6	+	+	+
075	Salmonella litchfield	55-3	+	+	+

	TABLE 7 - 4


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

076	Salmonella	55-4	+	+	+
	litchfield
077	Salmonella	55-6	+	+	+
	litchfield
078	Salmonella	55-7	+	+	+
	litchfield
079	Salmonella	55-8	+	+	+
	litchfield
080	Salmonella	55-12	+	+	+
	litchfield
081	Salmonella	55-13	+	+	+
	litchfield
082	Salmonella	61-2	+	+	+
	thompson
083	Salmonella	61-3	+	+	+
	thompson
084	Salmonella	61-4	+	+	+
	thompson
085	Salmonella	61-17	+	+	+
	thompson
086	Salmonella	61-18	+	+	+
	thompson
087	Salmonella	52-3	+	+	+
	thompson
088	Salmonella	52-4	+	+	+
	thompson
089	Salmonella	53-5	+	+	+
	thompson
090	Salmonella	53-6	+	+	+
	thompson
091	Salmonella	53-7	+	+	+
	thompson
092	Salmonella	53-20	+	+	+
	thompson
093	Salmonella	53-21	+	+	+
	thompson
094	Salmonella	NIAH1230	+	+	+
	thompson
095	Salmonella	59-36	+	+	+
	enteritidis
096	Salmonella	59-37	+	+	+
	enteritidis
097	Salmonella	59-38	+	+	+
	enteritidis
098	Salmonella	53-1	+	+	+
	enteritidis
099	Salmonella	53-2	+	+	+
	enteritidis
100	Salmonella	IFO3313	+	+	+
	enteritidis

	TABLE 7 - 5


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

101	Salmonella	61-19	+	+	+
	paratyphi B
102	Salmonella	61-20	+	+	+
	paratyphi B
103	Salmonella	61-21	+	+	+
	paratyphi B
104	Salmonella	63-1	+	+	+
	paratyphi B
105	Salmonella	63-2	+	+	+
	paratyphi B
106	Salmonella	63-3	+	+	+
	paratyphi B
107	Salmonella	58-55	+	+	+
	blockley
108	Salmonella	58-56	+	+	+
	blockley
109	Salmonella	58-57	+	+	+
	blockley
110	Salmonella	NIAH1197	+	+	+
	blockley
111	Salmonella	59-20	+	+	+
	infantis
112	Salmonella	59-21	+	+	+
	infantis
113	Salmonella	59-22	+	+	+
	infantis
114	Salmonella	NIAH1218	+	+	+
	infantis
115	Salmonella agona	59-1	+	+	+
116	Salmonella agona	59-2	+	+	+
117	Salmonella agona	59-3	+	+	+
118	Salmonella	58-7	+	+	+
	london
119	Salmonella	58-8	+	+	+
	london
120	Salmonella	58-9	+	+	+
	london
121	Salmonella	58-27	+	+	+
	senftenberg
122	Salmonella	58-28	+	+	+
	senftenberg
123	Salmonella	58-29	+	+	+
	senftenberg
124	Salmonella	57-1	+	+	+
	oranienburg
125	Salmonella	57-2	+	+	+
	oranienburg

	TABLE 7 - 6


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

126	Salmonella	54-4	+	+	+
	montevideo
127	Salmonella	NIAH1221	+	+	+
	montevideo
128	Salmonella	IFO3163	+	+	+
	gallinarum
129	Salmonella	NIAH1198	+	+	+
	choleraesuis
130	Salmonella derby	NIAH1199	+	+	+
131	Salmonella give	NIAH1214	+	+	+
132	Salmonella	56-44	+	+	+
	havana
133	Salmonella	NIAH1216	+	+	+
	heiderberg

[Experiment 2][0232]
To determine whether the results obtained in [0233] Experiment 1 are specific to the araC gene of Salmonella species, DNAs of clinically important diarrheal bacteria other than Salmonella species and other pathogenic bacteria are examined with the primers of the present invention. In particular, differentiation between Salmonella species and Citrobacter species, which has been difficult by conventional methods, is carefully examined.
The same procedure as used in [0234] Experiment 1 is followed, except for the procedure of preparation of specimens.
Preparation of Specimens [0235]
[0236] Clostridium perfringens, Campylobacter jejuni, Campylobacter coli, Bacteroides fragilis, Bacteroides vulgatus, Lactobacillus acidophilus and Bifidobacterium adolescentis are cultured at 37° C. under anaeorbic conditions, while Neisseria gonorrhoeae and Neisseria meningitidis are cultured in the presence of 3-10% CO₂.
Human placenta DNA, at a concentration of 1 μg/ml, is subjected to PCR in the same manner as above. [0237]
Results [0238]

Tables 8-1 to 8-3 shows the results of the test using the combinations of the primers of the present invention. These combinations of primers do not amplify any DNAs of bacterial strains other than Salmonella species or DNAs of human placenta. It is of particular importance that the combinations of the primers of the present invention do not amplify any DNAs of Citrobacter species which are closely akin to and hardly differentiated from Salmonella species. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the DNAs of Salmonella species, with high reliability.

	TABLE 8 - 1


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

01	Bacillus cereus	ATCC14579	−	−	−
02	Bacillus subtilis	JCM1465	−	−	−
03	Staphylococcus	JCM2413	−	−	−
	aureus
04	Staphylococcus	JCM2414	−	−	−
	epidermidis
05	Clostridium	ATCC12917	−	−	−
	perfringens
06	Vibrio cholerae	ATCC25872	−	−	−
07	Vibrio cholerae	ATCC9458	−	−	−
	type Ogawa
08	Vibrio cholerae	ATCC9459	−	−	−
	type Inaba
09	Vibrio cholerae	61H-151	−	−	−
10	Vibrio	WP-1	−	−	−
	parahaemolyticus
11	Vibrio fluvialis	JCM3752	−	−	−
12	Campylobacter	JCM2013	−	−	−
	jejuni
13	Campylobacter	JCM2529	−	−	−
	coli
14	Escherichia coli	JCM1649	−	−	−
15	Escherichia coli	H10407	−	−	−
16	Escherichia coli	WHO	3	−	−	−
17	Escherichia coli	WHO 47	−	−	−
18	Escherichia coli	T-1	−	−	−
19	Escherichia coli	T-40	−	−	−
20	Yersinia	ATCC9610	−	−	−
	enterocolitica
21	Shigella dysenteriae	ATCC9361	−	−	−
22	Shigella boydii	ATCC9210	−	−	−
23	Shigella flexneri	ATCC11836	−	−	−
24	Shigella sonnei	ATCC9290	−	−	−
25	Bacteroides flagilis	ATCC23745	−	−	−

	TABLE 8 - 2


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

26	Bacteroides vulgatus	JCM5826	−	−	−
27	Proteus vulgaris	JCM1668	−	−	−
28	Proteus mirabilis	ATCC29906	−	−	−
29	Streptococcus pyogenes	ATCC12344	−	−	−
30	Streptococcus pneumoniae	ATCC33400	−	−	−
31	Heamophilis influenzae	ATCC33391	−	−	−
32	Klebsiella pneumoniae	JCM1662	−	−	−
33	Neisseria gonorrbeae	ATCC19424	−	−	−
34	Neisseria meningitidis	ATCC13077	−	−	−
35	Listeria monocytogenes	ATCC15313	−	−	−
36	Lactobacillus acidophilus	JCM1132	−	−	−
37	Bifidobacterium adolescentis	JCM1275	−	−	−
38	Fusobacterium nucleatum	ATCC25586	−	−	−
39	Propionibacterium acnes	ATCC6919	−	−	−
40	Veillonella atypica	ATCC17744	−	−	−
41	Pseudomonas aeruginosa	IFO12689	−	−	−
42	Corynebacterium diphtheriae	JCM1310	−	−	−
43	Peptostreptococcus anaerobius	ATCC27337	−	−	−
44	Citrobacter freundii	ATCC6750	−	−	−
45	Citrobacter freundii	ATCC6879	−	−	−
46	Citrobacter freundii	ATCC8090	−	−	−
47	Citrobacter freundii	ATCC8454	−	−	−
48	Citrobacter freundii	ATCC10053	−	−	−
49	Citrobacter freundii	ATCC10625	−	−	−
50	Citrobacter freundii	ATCC10787	−	−	−

	TABLE 8 - 3


	Combination of primers*

No	Strains	7 + 8**	9 + 10**	11 + 8**

51	Citrobacter freundii	ATCC11102	−	−	−
52	Citrobacter freundii	ATCC11811	−	−	−
53	Citrobacter freundii	ATCC29063	−	−	−
54	Citrobacter freundii	ATCC29219	−	−	−
55	Citrobacter freundii	ATCC29220	−	−	−
56	Citrobacter freundii	ATCC29221	−	−	−
57	Citrobacter freundii	ATCC29222	−	−	−
58	Citrobacter freundii	ATCC29935	−	−	−
59	Citrobacter freundii	ATCC33128	−	−	−
60	Citrobacter	ATCC25405	−	−	−
	amalonaticus
64	Citrobacter	ATCC25406	−	−	−
	amalonaticus
64	Citrobacter	ATCC25407	−	−	−
	amalonaticus
65	Citrobacter diversus	ATCC27156	−	−	−
65	Citrobacter diversus	ATCC29223	−	−	−
65	Citrobacter diversus	ATCC29224	−	−	−
66	Citrobacter diversus	ATCC29225	−	−	−
67	Citrobacter diversus	ATCC29936	−	−	−

Example 5

Detection of EHEC (VTEC) having the VT1 Gene

[Experiment 1][0242]
Preparation of Specimens [0243]
The same procedure as used in Example 1 is followed except that 320 strains of EHEC (VTEC) strains. [0244]
Synthesis of primers [0245]
As primers for amplifying the VT1 gene of EHEC (VTEC), the above-described oligonucleotides SEQ ID NO: 12 and SEQ ID NO: 13 are selected based upon the known base sequence of the VT1 gene [Takao T., et al., Microb. Pathog., 5, 357-369(1988)]. These oligonucleotides are chemically synthesized by the same method as in [0246] Experiment 1 of Example 1.
PCR [0247]
PCR is carried out under the same reaction conditions as in Example 1 except that the following oligonucleotide combination is used: [0248]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 12+Oligonucleotide SEQ ID NO: 13[0249]
Detection [0250]
Agarose Gel Electrophoresis [0251]
The same procedure as in Example 1 is followed. [0252]
Colony Hybridization Test [0253]
A colony hybridization test is carried out using an oligonucleotide probe specific to the VT1 gene and that specific to the VT2 gene according to the procedure described by Grunstein [Grunstein, M. and Hogness, D., Proc. Natl. Acad. Sci., 72, 3961(1975)]. [0254]
Results [0255]
The base sequence of the VT1 gene of EHEC(VTEC) has already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when the oligonucleotides SEQ ID NO: 12 and SEQ ID NO: 13 of the present invention are used in combination, a nucleotide fragment of 349 bases (or a nucleotide duplex of 349 base pairs) should be amplified. When this estimation accords with the length of the amplified nucleotide fragment, it is judged that PCR using the combination of primers accurately amplifies the target region in the VT1 gene, and that the bacterial strain in the specimen has the VT1 gene. The results obtained from the agarose gel electrophoresis and from the colony hybridization test for 320 test strains are given in Table 9. Table 9 shows that PCR using the primers of the present invention amplifies only DNAs of the strains which give a positive result for the VT1 gene in the colony hybridization test, and that it does not amplify the DNA of the VT1 negative strains. This indicates that PCR using the primers of the present invention is capable of accurately amplifying the VT1 gene and that EHEC(VTEC) having the VT1 gene can be detected with high accuracy by using the oligonucleotides of the present invention. [0256]

TABLE 9

Accuracy of the primers

Results of colony hybridization test

Positive Negative

for both for both

Positive Positive VT 1 and VT 1 and

for for VT 2 VT 2

VT 1 gene VT 2 gene genes genes

Results Positive 39 0 53 0

of PCR Negative 0 185 0 43
[Experiment 2][0257]
To determine whether the results obtained in [0258] Experiment 1 are specific to EHEC (VTEC) having the VT1 gene, the DNAs of clinically important pathogenic bacteria other than EHEC (VTEC) are examined with the primers of the present invention. The same procedure as used in Experiment 1 is followed, except for the procedure of preparation of specimens.
Preparation of Specimens [0259]
Each strain listed in Table 10 is treated in the same manner as in [0260] Experiment 2 of Example 1. Among strains listed in Table 10, the following strains are cultured under anaerobic conditions: Clostridium perfringens, Campylobacter jejuni, Bacteroides fragills, Bacteroides vulgatus and Lactobacillus acidophilus.
Results [0261]
Table 10 shows the results from the test using the combinations of primers of the present invention. Although the combinations of primers do not amplify DNAs of any other strains than EHEC(VTEC) except for a certain type of Shigella species ([0262] Shigella dysenteriae type I).

It is well known that the differentiation between EHEC (VTEC) and Shigella dysenteriae is impossible because Shigella dysenteriae has the VT1 gene. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the DNAs of the bacteria having the VT1 gene.

TABLE 10


Reactivity with un-targeted gene

Combination of primers

No	Strains	12 + 13*	14 + 15*	16 + 18*	17 + 18*

1	Bacillus cereus	ATCC 14579	−	−	−	−
2	B. subtilis	JCM 1465	−	−	−	−
3	Staphylococcus aureus	JCM 2413	−	−	−	−
4	S. epidermidis	JCM 2414	−	−	−	−
5	Salmonella typhimurium	IFO 12529	−	−	−	−
6	S. enteritidis	IFO 3163	−	−	−	−
7	Clostridium perfringens	ATCC 12917	−	−	−	−
8	Vibrio fluvialis	JCM 3752	−	−	−	−
9	Campylobacter jejuni	JCM 2013	−	−	−	−
10	C. coli	JCM 2529	−	−	−	−
11	Escherichia coli	JCM 1649	−	−	−	−
12	Yersinia enterocolitica	ATCC 9610	−	−	−	−
13	Shigella dysenteriae	ATCC 9361	+	−	+	+
14	S. flexneri	ATCC 29903	−	−	−	−
15	S. sonnei	ATCC 29930	−	−	−	−
16	Bacteroides fragilis	ATCC 23745	−	−	−	−
17	B. vulgatus	JCM 5826	−	−	−	−
18	Enterococcus faecalis	JCM 5803	−	−	−	−
19	Klebsiella pneumoniae	JCM 1662	−	−	−	−
20	Proteus vulgaris	JCM 1668	−	−	−	−
21	Citrobacter freundii	ATCC 33128	−	−	−	−
22	Streptococcus pyogenes	ATCC 12344	−	−	−	−
23	S. pneumoniae	ATCC 33400	−	−	−	−
24	Haemophilus influenzae	ATCC 33391	−	−	−	−
25	Proteus mirabilis	ATCC 29906	−	−	−	−
26	Neisseria gonorrhoeae	ATCC 19424	−	−	−	−
27	N. meningitidis	ATCC 13077	−	−	−	−
28	Listeria monocytogenes	ATCC 15313	−	−	−	−
29	Lactobacillus acidophilus	JCM 1132	−	−	−	−
30	Bifidobacterium adolescentis	JCM 1275	−	−	−	−
31	Fusobacterium nucleatum	ATCC 2558	−	−	−	−
32	Propionibacterium acnes	ATCC 6919	−	−	−	−
33	Veillonella atypica	ATCC 17744	−	−	−	−
34	Pseudomonas aeruginosa	IFO 12689	−	−	−	−
35	Corynebacterium diphtheriae	JCM 1310	−	−	−	−
36	Peptostreptococcus anaerobius	ATCC 27337	−	−	−	−
37	Vibrio cholerae	ATCC 25872	−	−	−	−
38	V. cholerae type Ogawa	ATCC 9458	−	−	−	−
39	V. chalerae type Inaba	ATCC 9459	−	−	−	−

Example 6

Detection of EHEC (VTEC) having the VT2 Gene

[Experiment 1][0264]
Preparation of Specimens [0265]
The same procedure as used in [0266] Experiment 1 of Example 5 is followed.
Synthesis of Primers [0267]
As primers for amplifying the VT2 gene of EHEC (VTEC) strains, the above-described oligonucleotides SEQ ID NO: 14 and SEQ ID NO: 15 are selected based upon the known base sequence of the VT2 gene [Jackson, M. P., et al., FEMS Microbio. Lett., 44, 109-114(1987)]. These oligonucleotides are chemically synthesized by the same method as in Example 1. [0268]
PCR [0269]
PCR is carried out under the same reaction conditions as in Example 1 except that the following oligonucleotide combination is used:[0270]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 14+Oligonucleotide SEQ ID NO: 15 [0271]
Detection [0272]
Agarose Gel Electrophoresis [0273]
The same procedure as in Example 1 is followed. [0274]
Colony Hybridization Test [0275]
The same procedure as in [0276] Experiment 1 of Example 5 is followed.
Results [0277]
The base sequence of the VT2 gene of EHEC(VTEC) has already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when oligonucleotides SEQ ID NO: 14 and SEQ ID NO: 15 of the present invention are used in combination, a nucleotide fragment of 404 bases (or a nucleotide duplex of 404 base pairs) should be amplified. When this estimation accords with the length of the amplified nucleotide fragment, it is judged that PCR using the combination of primers accurately amplifies the target region in the VT2 gene, and that the bacterial strain in the specimen has the VT2 gene. The results obtained from the agarose gel electrophoresis with 320 test strains and from the colony hybridization test are given in Table 11. PCR using the primers of the present invention amplifies only DNAs of the strains which give a result positive for the VT2 gene in the colony hybridization test, showing no amplification of the DNA of the VT2 negative strains. This indicates that PCR using the primers of the present invention is capable of accurately amplifying the VT2 gene and that EHEC(VTEC) having the VT2 gene can be detected with high accuracy by using the oligonucleotides of the present invention. [0278]

TABLE 11

Accuracy of the primers

Results of colony hybridization test

Positive Negative

for both for both

Positive Positive VT 1 and VT 1 and

for for VT 2 VT 2

VT 1 gene VT 2 gene genes genes

Results Positive 0 185 53 0

of PCR Negative 39 0 0 43
[Experiment 2][0279]
To determine whether the results obtained in [0280] Experiment 1 are specific to EHEC (VTEC) having the VT2 gene, the DNAs of clinically important pathogenic bacteria other than EHEC (VTEC) are examined with the primers of the present invention. The same procedure as used in Experiment 2 of Example 5 is followed.
Results [0281]
Table 10 shows the results of the test using the combinations of primers of the present invention. All the combinations of primers in Table 10 do not amplify the DNAs of pathogenic bacteria other than EHEC (VTEC). It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the DNAs of the bacteria having the VT2 gene. [0282]

Example 7

Detection of EHEC (VTEC) having the VT1 Gene, the VT2 Gene or a Variant Gene of the VT2 Gene

[Experiment 1][0283]
Preparation of Specimens [0284]
The same procedure as used in [0285] Experiment 1 of Example 1 is followed.
Synthesis of Primers [0286]
As primers for amplifying the VT1 gene, the VT2 gene or a variant gene of VT2 (VT2vha, VT2vhb or VT2vp1), the above-described oligonucleotides SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 are selected. These oligonucleotides are chemically synthesized by the same method as in [0287] Experiment 1 of Example 1.
PCR [0288]
PCR is carried out under the same reaction conditions as in Example 1 except that any one of the following oligonucleotide combinations is used:[0289]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 16+Oligonucleotide SEQ ID NO: 18; and [0290]
Oligonucleotide SEQ ID NO: 17+Oligonucleotide SEQ ID NO: 18.[0291]
Detection [0292]
Agarose Gel Electrophoresis [0293]
The same procedure as in [0294] Experiment 1 of Example 1 is followed.
Colony Hybridization Test [0295]
The same procedures as in [0296] Experiment 1 of Example 1 are followed.
Results [0297]
The base sequences of the VT1 gene, the VT2 gene, the VT2vha gene, the VT2vhb gene and the VT2vp1 gene of EHEC(VTEC) have already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when the oligonucleotides SEQ ID NO: 16 and SEQ ID NO: 18 of the present invention are used in combination, a nucleotide fragment of 495 bases (or a nucleotide duplex of 495 base pairs) should be amplified. When this estimation accords with the length of the amplified nucleotide fragment, it is judged that PCR using the combination of primers accurately amplifies the target region in the VT1 gene, the VT2 gene, the VT2vha gene, the VT2vhb gene or the VT2vp1 gene, and that some bacterial strains in the specimen have any one of these genes. The results obtained from the agarose gel electrophoresis and from the colony hybridization test with 320 test strains are given in Table 12. PCR using the primers of the present invention amplifies only DNA of the strains which give the positive result for the VT1 gene or the VT2 gene, showing no amplification of the DNA of the strains negative for these genes. This indicates that PCR using the primers of the present invention is capable of accurately amplifying the VT1 gene or the VT2 gene (including its variant genes) and that EHEC(VTEC) having the VT1 gene or the VT2 gene or the both can be detected with high accuracy by using the oligonucleotides of the present invention. [0298]

TABLE 12

Accuracy of the primers

Results of colony

hybridization test

Positive Negative for

for VT1 or both VT1 and

VT2 gene VT2 genes

Results Combina- 16 + 18* Positive 277 0

of PCR tion Negative 0 43

of primers 17 + 18* Positive 277 0

Negative 0 43
[Experiment 2][0299]
To determine whether the results obtained in [0300] Experiment 1 are specific to EHEC (VTEC) having the VT1 gene or the VT2 gene, DNAs of clinically important pathogenic bacteria other than EHEC (VTEC) are examined with the primers of the present invention. The same procedure as used in Experiment 2 of Example 5 is followed.
Results [0301]
Table 10 shows the results from the test using the combinations of the primers of the present invention. PCR using the combinations of the primers does not amplify DNAs of any other strains than EHEC (VTEC) except for the DNA of a certain type of Shigella species ([0302] Shigella dysenteriae type I).
It is well known that differentiation between EHEC (VTEC) and [0303] Shigella dysenteriae is impossible only by detecting the VT1 gene because not only EHEC (VTEC) but also Shigella dysenteriae has the VT1 gene. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the DNAs of the bacteria having the VT1 gene or the VT2 gene.

Example 8

Detection of Staphylococcus aureus having the TSST-1 Gene

[Experiment 1][0304]
Preparation of Specimens [0305]
A total of 343 strains of [0306] Staphylococcus aureus are used. These strains are derived from food poisoning cases and the environment, and isolated from sources such as diarrheal stool, vomit and food. Each strain is inoculated to a brain heart infusion medium (manufactured by BBL Co., Ltd.), and subjected to overnight shaking culture at 37° C. under aerobic conditions. Each culture broth is diluted 10 folds with TE buffer, and heated at 95° C. for 5 minutes, followed by centrifugation at 5000 rpm for 1 minute; the supernatants are used as specimens.
Synthesis of Primers [0307]
As primers for amplifying the TSST-1 gene of [0308] Staphylococcus aureus, the above-described oligonucleotides SEQ ID NO: 19 to SEQ ID NO: 22 are selected based upon the known base sequences of the TSST-1 gene of Staphylococcus aureus [Blomster-Hautamaa et al., J. Biol. chem., 26, 15783-15786 (1986)], and chemically synthesized by the same method as in Experiment 1 of Example 1.
PCR [0309]
To 3 μl of the above-described specimen solution, 16.05 μl of sterile distilled water, 3 μl of 10×reaction buffer, 4.8 μl of dNTP solution, 1.0 μl of primer (1), 1.0 μl of primer (2), and 0.15 μl of a thermostable DNA polymerase are added to prepare 30 μl of a reaction mixture. This reaction mixture is overlaid with 50 μl of mineral oil (produced by SIGMA). The contents of the solutions used and the primers (1) and (2) are as follows: [0310]
10×reaction buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 15 mM MgCl[0311] ₂, 0.1% (w/v) gelatin.
dNTP solution: A mixture of dATP, dCTP, dGTP and dTTP, each having a final concentration of 1.25 mM. [0312]
Primers: Aqueous solution of the above-described chemically synthesized purified oligonucleotides (concentration, 3.75 OD/ml) is prepared. Any one of the following oligonucleotide combinations is used:[0313]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 20+oligonucleotide SEQ ID NO: 21 [0314]
Oligonucleotide SEQ ID NO: 19+Oligonucleotide SEQ ID NO: 22 and [0315]
Oligonucleotide SEQ ID NO: 20+Oligonucleotide SEQ ID NO: 22 [0316]
Thermostable DNA polymerase: Taq DNA polymerase (5 unit/ml; produced by Perkin Elmer Cetus). [0317]
The reaction conditions are as follows: [0318]
Thermal denaturation: 94° C. for 1 minute. [0319]
Annealing: 55° C. for 1 minute. [0320]
Polymerization: 72° C. for 1 minute. [0321]
The cycle of thermal denaturation, primer annealing and polymerization (5.7 minutes) is repeated for 35 cycles (entire time, about 3 hours). This procedure is performed using a DNA thermal cycler (produced by Perkin Elmer Cetus) in which the above reaction conditions are programmed. [0322]
Detection [0323]
Agarose Gel Electrophoresis [0324]
The same procedure as in Example 1 is followed. [0325]
Reversed Passive Latex Agglutination (RPLA) Test [0326]
A commercially available RPLA kit for detection of TSST-1 of [0327] Staphylococcus aureus (TST-RPLA “SEIKEN” produced by DENKA SEIKEN) is purchased. Specimens are prepared and tested according to the instruction manual attached except that the preparation of specimens is partially modified in order for the test strains to produce sufficient amount of its enterotoxin. That is, the brain heart infusion is changed to the one produced by BBL Co., Ltd.
Results [0328]
Table 13 shows the comparison of the results of the PCR method of the present invention with the results of the TST-RPLA which are conventionally used. The data indicates that the detection method of the present invention can detect the TSST-1 gene of [0329] Staphylococcus aureus with a sensitivity comparable to or higher than the conventional TST-RPLA method. The data in Table 13 show that 17 of the 18 PCR positive strains are also positive by RPLA, and that 325 strains are negative by both PCR and RPLA. That is, except one strain which is positive by PCR and negative by RPLA, the same results are obtained by PCR and by RPLA. The strain, for which the result by PCR and that by RPLA disaccord with each other, is tested by the Southern blot hybridization, and is confirmed to be positive for the TSST-1 gene.

TABLE 13

Comparison of PCR and TST-RPLA

PCR positive PCR negative

TST-RPLA positive 17 0

TST-RPLA negative 1 325
FIG. 1 shows the results of the sensitivity test for the PCR primers of the present invention by electrophoresis. In the figure, [0330] numerals 1 to 9 indicate the number of DNA copies used in the PCR reaction: 1 indicates 10⁷copies; 2, 10⁶copies; 3, 10⁵copies; 4, 10 ⁴copies; 5, 10³copies; 6, 10²copies; 7, 10 copies; 8, 1 copy; and 9, absence of DNA. From this figure, it is obvious that only 10 copies of DNA can be detected by the method of the present invention.
[Experiment 2][0331]
To determine whether the results obtained in [0332] Experiment 1 are specific to Staphylococcus aureus having the TSST-1 gene, DNAs of other clinically important pathogenic bacteria are examined with the primers of the present invention. The same procedure as used in Experiment 1 is followed, except for the method of preparation of specimens.
Preparation of Specimens [0333]
Each strain listed in Table 14 is treated in the same manner as in [0334] Experiment 2 of Example 5.
Results [0335]

Table 14 shows the results of the test using the primers of the present invention. PCR using the primers does not amplify any DNAs of other strains including those causative for food poisoning. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the DNA of Staphylococcus aureus having the TSST-1 gene. The similar results are obtained with the other combinations of the primers of the present invention which are not listed in Table 14.

	TABLE 14


	Combination of primers

No	Strains	20 + 21*	19 + 22*	20 + 22*

1	Bacillus cereus	ATCC 14579	−	−	−
2	Bacillus subtilis	JCM 1455	−	−	−
3	Staphylococcus aureus	JCM 2413	−	−	−
4	Staphylococcus epidermidis	JCM 2414	−	−	−
5	Salmonella typhimurium	IFO 12529	−	−	−
6	Salmonella enteritidis	IFO 3163	−	−	−
7	Clostridium perfringens	ATCC 12917	−	−	−
8	Vibrio cholerae	ATCC 25872	−	−	−
9	Vibrio cholerae type Ogawa	ATCC 9458	−	−	−
10	Vibrio cholerae type Inaba	ATCC 8459	−	−	−
11	Vibrio fluvialis	JCM 3752	−	−	−
12	Campylobacter jejuni	JCM 2013	−	−	−
13	Campylobacter coli	JCM 2529	−	−	−
14	Eschericia coli	JCM 1548	−	−	−
15	Yersinia enterocolitica	ATCC 5610	−	−	−
16	Shigella dysenteriae	ATCC 3361	−	−	−
17	Shigella flexneri	ATCC 29903	−	−	−
18	Shigella sonnei	ATCC 29930	−	−	−
19	Bacteroides fragilis	ATCC 23745	−	−	−
20	Bacteroides vulgatus	JCM 5826	−	−	−
21	Enterococcus faecalis	JCM 5803	−	−	−
22	Klebsiella pneumoniae	JCM 1662	−	−	−
23	Protaus vulgaris	JCM 1688	−	−	−
24	Citrobacter freundii	ATCC 33128	−	−	−
25	Streptococcus pyogenes	ATCC 12344	−	−	−
26	Streptococcus pneumoniae	ATCC 33400	−	−	−
27	Haemophilus influenzae	ATCC 33391	−	−	−
28	Proteus mirabilis	ATCC 29906	−	−	−
29	Neisseria gonorrhoeae	ATCC 19424	−	−	−
30	Neisseria meningitidis	ATCC 13077	−	−	−
31	Listeria monocytogenes	ATCC 15313	−	−	−
32	Lactobacillus acidophilus	JCM 1132	−	−	−
33	Bifidobacterium adolescentis	JCM 1275	−	−	−
34	Fusobacterium nucleatum	ATCC 25585	−	−	−
35	Propionibacierium acnes	ATCC 5918	−	−	−
36	Veillonella atypica	ATCC 17744	−	−	−
37	Pseudomonas aeruginosa	IFO 12689	−	−	−
38	Corynebacterium diphtheriae	JCM 1310	−	−	−
39	Peptostreptococcus anaerobius	ATCC 27337	−	−	−

Example 9

Detection of Vibrio cholerae having the ctx Gene

[Experiment 1][0337]
Preparation of Specimens [0338]
The same procedure as used in Example 1 is followed except that 622 strains of [0339] Vibrio cholerae are used. These strains are isolated from patients with cholera, marine products (shrimp, snapping turtle), water collected from river, harbor, etc. Serotype, biotype, the numbers of the strains are listed in Table 15.

TABLE 15

Type and sources of Vibrio cholerae

Sources

Enviroment

Serotype Biotype Patients Food water Total

O1 Ogawa E1 Tor 148 125 71 344

Inaba 16 27 26 69

Ogawa Asia 15 0 0 15

Inaba (classical) 26 0 0 26

non O1 — 168 0 0 168

Total 373 152 97 622
Synthesis of Primers [0340]
As primers for amplifying the ctx gene of [0341] Vibrio cholerae, the above-described oligonucleotides SEQ ID NO: 23 to SEQ ID NO: 26 are selected based upon the known base sequences of the ctx gene of Vibrio cholerae [Lockman, H. and J. B. Kaper: J. Biol. Chem., 258, 13722-13726 (1983)], and chemically synthesized by the same method as in Experiment 1 of Example 1.
PCR [0342]
PCR is carried out under the same reaction conditions as in Example 1 except that any one of the following oligonucleotide combinations is used:[0343]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 23+Oligonucleotide SEQ ID NO: 25; and [0344]
Oligonucleotide SEQ ID NO: 24+Oligonucleotide SEQ ID NO: 26. [0345]
Detection [0346]
Agarose Gel Electrophoresis [0347]
To detect the amplified nucleotide fragments in the reaction solution, agarose gel electrophoresis is carried out by the same procedure as in Example 1. [0348]
Colony Hybridization Test [0349]
A colony hybridization test is carried out using an polynucleotide probe specific to the ctx gene [Kaper, J. B., J. G. Morris, Jr., and N. Nishibuchi (1988), DNA probes for pathogenic Vibrio species, 65-77. In F. C. Tenover (ed.), DNA probes for infectious diseases. CRC Press, Inc., Boca Raton, Fla.] according to the procedure described by Grunstein [Grunstein, M. and Hogness, D., Proc. Natl. Acad. Sci., 72, 3961(1975)]. [0350]
Results [0351]
The base sequences of the ctx gene of [0352] Vibrio cholerae have already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when the oligonucleotides SEQ ID NO: 23 and SEQ ID NO: 25 of the present invention are used in combination, a nucleotide fragment of 169 bases (or a nucleotide duplex of 169 base pairs) should be amplified. The combination of SEQ ID NO: 24 and SEQ ID NO: 26 should amplify a nucleotide fragment of 307 bases (or a nucleotide duplex of 307 base pairs). When the estimated length of nucleotide accords with the length of the amplified nucleotide fragments, it is judged that PCR using the combination of primers accurately amplifies the target region in the ctx gene, and that the bacterial strain in the specimen has the ctx gene. The results obtained from the agarose gel electrophoresis and from the colony hybridization test with 662 test strains are given in Table 16. PCR using the primers of the present invention amplifies only DNAs of the strains which give a result positive for the ctx gene in the colony hybridization test, showing no amplification of the DNA of the ctx gene negative strains. This indicates that PCR using the primers of the present invention is capable of accurately amplifying the ctx gene and that Vibrio cholerae having the ctx gene can be detected with high accuracy by using the oligonucleotides of the present invention. Table 16 shows the result obtained with oligonucleotides SEQ ID NO: 24 and SEQ ID NO: 26. The combination of SEQ ID NO: 23 and SEQ ID NO: 25 also gives a similar result.

TABLE 16

Accuracy of primer combination of SEQ ID NO: 24 and

SEQ ID NO: 26.

Results of colony hybridization test

ctx gene positive ctx gene negative

Results positive 412 0

of PCR negative 0 210
FIG. 2 shows that PCR using the combinations of the primers of the present invention can accurately detect the ctx gene irrespective of the source, serological type and biological type of the strains. Heat extracts of the following strains are used as the template DNA solutions:[0353]
[0354] Lanes 1 to 3: Vibrio cholerae (El Tor—Ogawa type, the ctx gene positive strain)
[0355] Lanes 4 to 6: Vibrio cholerae (El Tor—Inaba type, the ctx gene positive strain)
Lane 7: [0356] Vibrio cholerae (Classical—Ogawa type, the ctx gene positive strain)
Lane 8: [0357] Vibrio cholerae (Classical—Inaba type, the ctx gene positive strain)
[0358] Lanes 9 to 10: Vibrio cholerae (non-01, the ctx gene positive strain)
Lane 11: [0359] Vibrio cholerae (El Tor—Ogawa type, the ctx gene negative strain)
Lane 12: [0360] Vibrio cholerae (El Tor—Inaba type, the ctx gene negative strain)
Lane 13: Enterotoxigenic [0361] Escherichia Coli (Thermolabile enterotoxin gene positive strain)
[Experiment 2][0362]
To determine whether the results obtained in [0363] Experiment 1 are specific to Vibrio cholerae having the ctx gene, the genes of other clinically important pathogenic bacteria are examined with the method of the present invention. The same procedure as used in Experiment 1 is followed, except for the method of preparation of specimens.
Preparation of Specimens [0364]
Each strain listed in Table 17 is treated in the same manner as in [0365] Experiment 2 of Example 5.
Results [0366]

Table 17 shows the results of the test using a combination of primers of the present invention. PCR using the primers does not amplify DNAs of any other pathogenic strains tested. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the DNAs of Vibrio cholerae having the ctx gene. Similar results are obtained also for the other combination of primers of the present invention which is not listed in Table 17.

TABLE 17


Reactivity with DNA of bacteria other than
Vibrio cholerae

No	S t r a i n s	+/−*

1	Bacillus cereus ATCC 14579	−
2	B. subtilis JCM 1465	−
3	Staphylococcus aureus JCM 2413	−
4	S. epidermidis JCM 2414	−
5	Salmonella typhimurium IFO 12529	−
6	S. enteritidis IFO 3163	−
7	Clostridium perfringens ATCC 12917	−
8	Vibrio fluvialis JCM 3752	−
9	Campylobacter jejuni JCM 2013	−
10	C. coli JCM 2529	−
11	Escherichia coli JCM 1649	−
12	Yersinia enterocolitica ATCC 9610	−
13	Shigella dysenteriae ATCC 9361	−
14	S. flexneri ATCC 29903	−
15	S. sonnei ATCC 29930	−
16	Bacteroides fragilis ATCC 23745	−
17	B. vulgatus JCM 5826	−
18	Enterococcus faecalis JCM 5803	−
19	Klebsiella pneumoniae JCM 1662	−
20	Proteus vulgaris JCM 1668	−
21	Citrobacter freundii ATCC 33128	−
22	Streptococcus pyogenes ATCC 12344	−
23	S. pneumoniae ATCC 33400	−
24	Haemophilus influenzae ATCC 33391	−
25	Proteus mirabilis ATCC 29906	−
26	Neisseria gonorrhoeae ATCC 19424	−
27	N. meningitidis ATCC 13077	−
28	Listeria monocytogenes ATCC 15313	−
29	Lactobacillus acidophilus JCM 1132	−
30	Bifidobacterium adolescentis JCM 1275	−
31	Fusobacterium nucleatum ATCC 2558	−
32	Propionibacterium acnes ATCC 6919	−
33	Veillonella atypica ATCC 17744	−
34	Pseudomonas aeruginosa IFO 12689	−
35	Corynebacterium diphtheriae JCM 1310	−
36	Peptostreptococcus anaerobius ATCC 27337	−
37	Human placental DNA

Example 10

Detection of Clostridium perfringens having the Enterotoxin Gene

[Experiment 1][0368]
Preparation of Specimens [0369]
The strains of [0370] Clostridium perfringens used are 11 strains isolated from patients, and provided by institutes where each strain is stored. Each strain is inoculated to GAM broth (manufactured by Nissui Pharmaceutical Co., Ltd.) and subjected to overnight shaking culture at 37° C. under anaerobic conditions. Each culture broth is diluted 10 folds with 10 mM Tris-HCl buffer, pH 7.5, and heated at 95° C. for 10 minutes, followed by centrifugation to use the supernatant as a specimen.
Synthesis of Primers [0371]
As primers for amplifying the enterotoxin gene of [0372] Clostridium perfringens, the above-described oligonucleotides SEQ ID NO: 27 to SEQ ID NO: 35 are selected based upon the known base sequences of the enterotoxin gene of Clostridium perfringens [Maruke van Damme-Jongsten, Antonie van Leeuwenhoek, 56, 181-190(1989)], and chemically synthesized by the same method as in Experiment 1 of Example 1.
PCR [0373]
PCR is carried out under the same reaction conditions as in Example 1 except that any one of the following oligonucleotide combinations is used: [0374]
Primer (1)+primer (2)=Oligonucleotide SEQ ID NO: 27+Oligonucleotide SEQ ID NO: 32; [0375]
Oligonucleotide SEQ,ID NO: 28+Oligonucleotide SEQ ID NO: 33; [0376]
Oligonucleotide SEQ ID NO: 29+Oligonucleotide SEQ ID NO: 33; [0377]
Oligonucleotide SEQ ID NO: 30+Oligonucleotide SEQ ID NO: 34; and [0378]
Oligonucleotide SEQ ID NO: 31+Oligonucleotide SEQ ID NO: 35. [0379]
Detection [0380]
Agarose Gel Electrophoresis [0381]
To detect the amplified nucleotide fragments in the reaction solution, agarose gel electrophoresis is carried out by the same procedure as in Example 1. [0382]
FIG. 3 shows a part of the electrophoretic results. The upper part of the figure shows the results with Oligonucleotide SEQ ID NO: 28+Oligonucleotide SEQ ID NO: 33; and the lower part, the results with Oligonucleotide SEQ ID NO: 29+Oligonucleotide SEQ ID NO: 33. In the figure, M indicates the molecular weight marker; and [0383] lanes 1 to 13 respectively indicate ATCC 12925(lane 1), ATCC 12924(lane 2), ATCC 12922(lane 3), ATCC 12920(lane 4), ATCC 12916(lane 5), ATCC 12915(lane 6), ATCC 12918(lane 7), ATCC 12919(lane 8), ATCC 12921(lane 9), JCM 1296(lane 10), JCM 1416(lane 11), JCM 1382(lane 12), and TE (negative control, lane 13).
Southern Blot Hybridization Test [0384]
A southern blot hybridization test is carried out using an oligonucleotide probe specific to the enterotoxin gene of [0385] Clostridium perfringens according to the method described by Tada et al. [Tada, J. et al. Mol. Cell. Probe., 6, 477 (1992)].
Reversed Passive Latex Agglutination (RPLA) Test [0386]
A commercially available RPLA kit for detection of [0387] Clostridium perfringens enterotoxin (PET-RPLA “SEIKEN” produced by DENKA SEIKEN) is purchased. Specimens are prepared and tested according to the instruction manual attached.
Results [0388]

The base sequences of the enterotoxin gene of Clostridium perfringens have already been determined. Therefore, the length of the nucleotide fragments amplified by PCR using the oligonucleotides of the present invention as primers can easily be estimated. Specifically, when the oligonucleotides SEQ ID NO: 27 and SEQ ID NO: 32 of the present invention are used in combination, a nucleotide fragment of 473 bases (or a nucleotide duplex of 473 base pairs) should be amplified. When the estimated length accords with the length of the amplified nucleotide fragment, it is judged that PCR using the combination of the primers accurately amplifies the target region in the enterotoxin gene, and that the bacterial strain in the specimen has the enterotoxin gene. The results obtained from the agarose gel electrophoresis and from the RPLA test with the 11 test strains are given in Table 18.

	TABLE 18


	Combination of primers and length of amplified
	DNA (No. of b. p.)*

	Results	27 + 32**	28 + 33**	29 + 33**	30 + 34**	31 + 35**
Strains	of RPLA	473	456	421	267	156

01	Clostridium perfringens	ATCC 12915	+***	+	+	+	+	+
02	Clostridium perfringens	ATCC 12916	+	+	+	+	+	+
03	Clostridium perfringens	ATCC 12917	+	+	+	+	+	+
04	Clostridium perfringens	ATCC 12918	+	+	+	+	+	+
05	Clostridium perfringens	ATCC 12919	−	−	−	−	−	−
06	Clostridium perfringens	ATCC 12920	+	+	+	+	+	+
07	Clostridium perfringens	ATCC 12921	−	−	−	−	−	−
08	Clostridium perfringens	ATCC 12922	+	+	+	+	+	+
09	Clostridium perfringens	ATCC 12924	+	+	+	+	+	+
10	Clostridium perfringens	ATCC 12925	+	+	+	+	+	+
11	Clostridium perfringens	JCM 3816	−	−	−	−	−	−

In the Southern blot hybridization test, it is confirmed that the nucleotide fragments amplified with a combination of the primers of the present invention is a part of the enterotoxin gene sequences. The results are shown in FIG. 4. FIG. 4 corresponds to FIG. 3. In the figure, M indicates the molecular weight marker; and [0390] lanes 1 to 13 respectively indicate ATCC 12925(lane 1), ATCC 12924(lane 2), ATCC 12922(lane 3), ATCC 12920(lane 4), ATCC 12916(lane 5), ATCC 12915(lane 6), ATCC 12918(lane 7), ATCC 12919(lane 8), ATCC 12921(lane 9), JCM 1296(lane 10), JCM 1416(lane 11), JCM 1382(lane 12), and TE (negative control, lane 13).
These results indicate that PCR using the primers of the present invention is capable of accurately amplifying the enterotoxin gene in PCR and that [0391] Clostridium perfringens having the enterotoxin gene can be detected with high accuracy by using the oligonucleotides of the present invention.
[Experiment 2][0392]
To determine whether the results obtained in [0393] Experiment 1 are specific to Clostridium perfringens having the enterotoxin gene, the reactivity of the primers of the present invention with the DNAs of other Clostridium species and other clinically important pathogenic bacteria is examined. The same procedure as used in Experiment 1 is followed, except for the method of preparation of specimens.
Preparation of Specimens [0394]
Each strain listed in Tables 19 and 20 is treated in the same manner as in [0395] Experiment 2 of Example 5.
Results [0396]

Tables 19 and 20 show the results of the test using some of the combinations of primers of the present invention. All the combinations of the primers listed in the tables do not show any amplification of DNAs of other strains including pathogenic strains in PCR. It can therefore be concluded that the oligonucleotide primers of the present invention selectively react with the enterotoxin gene of Clostridium perfringens.

	TABLE 19


	Combination of primers and length of amplified
	DNA (No. of b. p.)*

	27 + 32**	28 + 33**	29 + 33**	30 + 34**	31 ± 35**
Strains	473	456	421	267	156

01	Clostridium absonum	ATCC 27555	−	−	−	−	−
02	Clostridium barati	JCM 1382	−	−	−	−	−
03	Clostridium bifermentans	ATCC 638	−	−	−	−	−
04	Clostridium butyricum	JCM 1391	−	−	−	−	−
05	Clostridium difficile	JCM 1296	−	−	−	−	−
06	Clostridium fallax	JCM 1398	−	−	−	−	−
07	Clostridium histolyticum	JCM 1403	−	−	−	−	−
08	Clostridium novyi	JCM 1406	−	−	−	−	−
09	Clostridium sordellii	JCM 3814	−	−	−	−	−
10	Clostridium sphenoides	JCM 1415	−	−	−	−	−
11	Clostridium spiroforme	JCM 1432	−	−	−	−	−
12	Clostridium sporogenes	JCM 1416	−	−	−	−	−
13	Clostridium tertiium	JCM 6289	−	−	−	−	−

	TABLE 20


	Combination of primers and length of amplified
	DNA (No. of b. p.)*

	27 + 32**	28 + 33**	29 + 33**	30 + 34**	31 + 35**
Strains	473	456	421	267	156

01	Vibrio cholerae	ATCC 25872	−	−	−	−	−
02	Vibrio cholerae type Ogawa	ATCC 9458	−	−	−	−	−
03	Vibrio cholerae type Inaba	ATCC 9459	−	−	−	−	−
04	Vibrio fluvialis	JCM 3752	−	−	−	−	−
05	Vibrio metschnikovii	ATCC 7708	−	−	−	−	−
06	Vibrio mimicus	ATCC 33653	−	−	−	−	−
07	Bacillus cereus	ATCC 14579	−	−	−	−	−
08	Bacillus subtilis	JCM 1465	−	−	−	−	−
09	Staphylococcus aureus	JCM 2413	−	−	−	−	−
10	Staphylococcus epidermidis	JCM 2414	−	−	−	−	−
11	Salmonella typhimurium	IFO 12529	−	−	−	−	−
12	Salmonella enteritidis	IFO 3163	−	−	−	−	−
13	Campylobacter jejuni	JCM 2013	−	−	−	−	−
14	Campylobacter coli	JCM 2529	−	−	−	−	−
15	Escherichia coli	JCM 1649	−	−	−	−	−
16	Yersinia enterocolitica	ATCC 9610	−	−	−	−	−
17	Shigella dysenteriae	ATCC 9361	−	−	−	−	−
18	Shigella flexneri	ATCC 29903	−	−	−	−	−
19	Shigella sonnei	ATCC 29930	−	−	−	−	−
20	Bacteroides fragilis	ATCC 23745	−	−	−	−	−
21	Bacteroides vulgatus	JCM 5826	−	−	−	−	−
22	Enterococcus faecalis	JCM 5803	−	−	−	−	−
23	Klebsiella pneumoniae	JCM 1662	−	−	−	−	−
24	Proteus mirabilis	ATCC 29906	−	−	−	−	−
25	Proteus vulgaris	JCM 1668	−	−	−	−	−
26	Citrobacter freundii	ATCC 33128	−	−	−	−	−
27	Streptococcus pyogenes	ATCC 12344	−	−	−	−	−
28	Streptococcus pneumoniae	ATCC 33400	−	−	−	−	−
29	Haemophilus influenzae	ATCC 33391	−	−	−	−	−
30	Neisseria gonorrhoeae	ATCC 19424	−	−	−	−	−
31	Neisseria meningitidis	ATCC 13077	−	−	−	−	−
32	Listeria monocytogenes	ATCC 15313	−	−	−	−	−
33	Lactobacillus acidophilus	JCM 1132	−	−	−	−	−
34	Bifidobacterium adolescentis	JCM 1275	−	−	−	−	−
35	Fusobacterium nucleatum	ATCC 25586	−	−	−	−	−
36	Propionibacterium acnes	ATCC 6919	−	−	−	−	−
37	Veillonella atypica	ATCC 17744	−	−	−	−	−
38	Pseudomonas aeruginosa	IFO 12689	−	−	−	−	−
39	Corynebacterium diphtheriae	JCM 1310	−	−	−	−	−
40	Peptostreptococcus anaerobius	ATCC 27337	−	−	−	−	−

The agarose gel electrophoresis used in the above examples of the present invention can differentiate nucleotide fragments from one another which are different in length by 5-10 bases (base pairs) for nucleotide fragments of not more than 100 bases (base pairs), and by 10-20 bases (base pairs) for nucleotide fragments of 100-500 bases (base pairs). In addition, the use of other gel material such as acrylamide makes it possible to improve the precision in measuring the length of nucleotide fragment. Thus, the reliability of the selective detection of the target gene in the present invention can further be increased. [0399]
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. [0400]

0

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 35

<210> SEQ ID NO 1

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Shigella dysenteriae

<400> SEQUENCE: 1

caacactgga tgatctcag 19

<210> SEQ ID NO 2

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Shigella dysenteriae

<400> SEQUENCE: 2

ccccctcaac tgctaata 18

<210> SEQ ID NO 3

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Shigella dysenteriae, Shigella flexneri, Shigella

boydii, Shigella sonnei

<400> SEQUENCE: 3

tgtatcacag atatggcatg c 21

<210> SEQ ID NO 4

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Shigella dysenteriae, Shigella flexneri, Shigella

boydii, Shigella sonnei

<400> SEQUENCE: 4

tccggagatt gttccatgtg 20

<210> SEQ ID NO 5

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Shigella dysenteriae, Shigella flexneri, Shigella

boydii, Shigella sonnei

<400> SEQUENCE: 5

caagatttaa ccttcgtcaa cc 22

<210> SEQ ID NO 6

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Shigella dysenteriae, Shigella flexneri, Shigella

boydii, Shigella sonnei

<400> SEQUENCE: 6

agttctcgga tgctatgctc 20

<210> SEQ ID NO 7

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Salmonella spp.

<400> SEQUENCE: 7

cgcggagagg gcgtcatt 18

<210> SEQ ID NO 8

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Salmonella spp.

<400> SEQUENCE: 8

gcaacgactc attaattacc g 21

<210> SEQ ID NO 9

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Salmonella spp.

<400> SEQUENCE: 9

atctggtcgc cgggctga 18

<210> SEQ ID NO 10

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Salmonella spp.

<400> SEQUENCE: 10

gcatcgcgca cacggcta 18

<210> SEQ ID NO 11

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Salmonella spp.

<400> SEQUENCE: 11

ggcgagcagt ttgtctgtc 19

<210> SEQ ID NO 12

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 12

caacactgga tgatctcag 19

<210> SEQ ID NO 13

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 13

ccccctcaac tgctaata 18

<210> SEQ ID NO 14

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 14

atcagtcgtc actcactggt 20

<210> SEQ ID NO 15

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 15

ccagttatct gacattctg 19

<210> SEQ ID NO 16

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 16

agtttacgtt agacttttcg ac 22

<210> SEQ ID NO 17

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 17

cggacagtag ttataccac 19

<210> SEQ ID NO 18

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Escherichia coli

<400> SEQUENCE: 18

ctgctgtcac agtgacaaa 19

<210> SEQ ID NO 19

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Staphylococcus aureus

<400> SEQUENCE: 19

cctttaaaag ttaaggttca tg 22

<210> SEQ ID NO 20

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Staphylococcus aureus

<400> SEQUENCE: 20

ggccaaagtt cgataaaaaa c 21

<210> SEQ ID NO 21

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Staphylococcus aureus

<400> SEQUENCE: 21

atttataggt ggtttttcag tat 23

<210> SEQ ID NO 22

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Staphylococcus aureus

<400> SEQUENCE: 22

ctgcttctat agtttttatt tca 23

<210> SEQ ID NO 23

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Vibrio cholerae

<400> SEQUENCE: 23

tgatgaaata aagcagtcag gt 22

<210> SEQ ID NO 24

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Vibrio cholerae

<400> SEQUENCE: 24

acagagtgag tactttgacc 20

<210> SEQ ID NO 25

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Vibrio cholerae

<400> SEQUENCE: 25

ggcacttctc aaactaattg ag 22

<210> SEQ ID NO 26

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Vibrio cholerae

<400> SEQUENCE: 26

ataccatcca tatatttggg ag 22

<210> SEQ ID NO 27

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 27

tctgaggatt taaaaacacc 20

<210> SEQ ID NO 28

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 28

accctcagta ggttcaagtc 20

<210> SEQ ID NO 29

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 29

atgaaacagg tacctttagc c 21

<210> SEQ ID NO 30

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 30

ggtaatatct ctgatgatgg at 22

<210> SEQ ID NO 31

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 31

taactcatac ccttggactc 20

<210> SEQ ID NO 32

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 32

gaaccttgat caatatttcc 20

<210> SEQ ID NO 33

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 33

gtagcagcag ctaaatcaag g 21

<210> SEQ ID NO 34

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 34

agtccaaggg tatgagttag 20

<210> SEQ ID NO 35

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Clostridium perfringens

<400> SEQUENCE: 35

ccatcaccta aggactgttc 20

Claims

What is claimed is:

1. A synthetic oligonucleotide of 10 to 30 bases which is complementary to a nucleotide sequence of a gene selected from the group consisting of the Shiga toxin gene of Shigella species, the ipaH gene of Shigella species and EIEC, the invE gene of Shigella species and EIEC, the arcC gene of Salmonella species, the toxic shock syndrome toxin-1 gene of Staphylococcus aureaus, the ctx gene of Vibrio cholerae, and the enterotoxin gene of Clostridium perfringens.

2. A synthetic oligonucleotide comprising a nucleotide sequence complementary to the synthetic oligonucleotide of claim 1.

3. A method for detecting a bacterial strain selected from the group consisting of Shigella species, EIEC, Salmonella species,Staphylococcus aureus, vibrio cholerae, and Clostridium perfringens, wherein the method comprises

(1) hybridizing one primer to a single-stranded target DNA as a template DNA present in a specimen and carrying out a primer extension reaction to give a primer extension product;

(2) denaturing the resulting DNA duplex to separate the primer extension product from the template DNA, the primer extension product functioning as the other template DNA for the other primer;

(3) repeating a cycle of simultaneous primer extension reaction with the two primers, separation of the primer extension products from the templates, and hybridization of primers to amplify a region of the target DNA, in the steps from (1) to (3) said primers being selected from the group consisting of oligonucleotides of claim 1 and a synthetic oligonucleotide comprising a nucleotide sequence complementary to the synthetic oligonucleotide; and

(4) detecting the amplified nucleotide sequence to determine whether a suspected bacterial strain is present in the specimen.

4. A kit for detection of a bacterial strain comprising at least a pair of primers selected from the group consisting of oligonucleotides of claims 1, and synthetic oligonucleotides comprising sequences complementary to the oligonucleotides of claim 1, a thermostable DNA polymerase, and dNTP solutions.

5. The synthetic oligonucleotide according to claim 1, wherein said gene is the Shiga toxin gene of Shigella dysenteriae type 1, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 1 or oligonucleotide SEQ ID NO: 2.

6. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to a synthetic oligonucleotide of 10 to 30 bases which in turn is complementary to a nucleotide sequence of a Shiga toxin gene of Shigella dysenteriae type 1, and wherein the synthetic oligonucleotide comprises bases complementary to at least 10 consecutive bases of oligonucleotide SEQ ID NO: 1 or oligonucleotide SEQ ID NO: 2.

7. The synthetic oligonucleotide according to claim 1, wherein said gene is the ipaH gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 3 or oligonucleotide SEQ ID NO: 4.

8. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to a synthetic oligonucleotide of 10 to 30 bases which in turn is complementary to a nucleotide sequence of an ipaH gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises bases complementary to at least 10 consecutive bases of oligonucleotide SEQ ID NO: 3 or oligonucleotide SEQ ID NO: 4.

9. The synthetic oligonucleotide according to claim 1, wherein said gene is the invE gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 5 or oligonucleotide SEQ ID NO: 6.

10. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to a synthetic oligonucleotide of 10 to 30 bases which in turn is complementary to a nucleotide sequence of an invE gene of Shigella species and EIEC, and wherein the synthetic oligonucleotide comprises bases complementary to at least 10 consecutive bases of oligonucleotide SEQ ID NO: 5 or oligonucleotide SEQ ID NO: 6.

11. The method according to claim 3, wherein said target DNA is a gene of Shigella species and EIEC and said primers are selected from the group consisting of oligonucleotides of claims 5 to 10.

12. The kit according to claim 4, wherein said pair of primers are selected from the group consisting of oligonucleotides of claims 5 to 10 for detection of Shigella species and EIEC.

13. The synthetic oligonucleotide according to claim 1, wherein said gene is the araC gene of Salmonella species, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 7, oligonucleotide SEQ ID NO: 8, oligonucleotide SEQ ID NO: 9, oligonucleotide SEQ ID NO: 10, and oligonucleotide SEQ ID NO: 11.

14. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of claim 13.

15. The method according to claim 3, wherein said target DNA is a gene of a Salmonella species and said primers are selected from the group consisting of oligonucleotides comprising at least 10 consecutive bases of the oligonucleotide of SEQ ID NO: 7, the oligonucleotide of SEQ ID NO: 8, the oligonucleotide of SEQ ID NO: 9, the oligonucleotide SEQ ID NO: 10, the oligonucleotide of SEQ ID NO: 11, the oligonucleotide complementary to SEQ ID NO: 7, the oligonucleotide complementary to SEQ ID NO: 8, the oligonucleotide complementary to SEQ ID NO: 9, the oligonucleotide complementary to SEQ ID NO: 10 and the oligonucleotide complementary to SEQ ID NO: 11.

16. The method according to claim 15, wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 7 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 8; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 9 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 10; and a combination in which one comprises at least 10 consecutive bases of Oligonucleotide SEQ ID NO: 11 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 8.

17. The kit according to claim 4, wherein said pair of primers is selected from the group consisting of oligonucleotides comprising at least 10 consecutive bases of the oligonucleotide of SEQ ID NO: 7, the oligonucleotide of SEQ ID NO: 8, the oligonucleotide of SEQ ID NO: 9, the oligonucleotide SEQ ID NO: 10, the oligonucleotide of SEQ ID NO: 11, the oligonucleotide complementary to SEQ ID NO: 7, the oligonucleotide complementary to SEQ ID NO: 8, the oligonucleotide complementary to SEQ ID NO: 9, the oligonucleotide complementary to SEQ ID NO: 10 and the oligonucleotide complementary to SEQ ID NO: 11.

18. The synthetic oligonucleotide according to claim 1, wherein said gene is the toxic shock syndrome toxin-1 gene of Staphylococcus aureus, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 19, oligonucleotide SEQ ID NO: 20, oligonucleotide SEQ ID NO: 21, and Oligonucleotide SEQ ID NO: 22.

19. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of claim 18.

20. The method according to claim 3, wherein said target DNA is a gene of Staphylococcus aureus and said primers are selected from the group consisting of oligonucleotides of claims 18 and 19.

21. The method according to claim 20, wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 20 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 21; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 19 and the other comprises at least TO consecutive bases of oligonucleotide SEQ ID NO: 22; and a combination in which one comprises at least 10 consecutive bases of Oligonucleotide SEQ ID NO: 20 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 22.

22. The kit according to claim 4, wherein said pair of primers are selected from the group consisting of oligonucleotides of claims 18 and 19 for detection of Staphylococcus aureus.

23. The synthetic oligonucleotide according to claim 1, wherein said gene is the cholera toxin gene of Vibrio cholerae, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 23, oligonucleotide SEQ ID NO: 24, oligonucleotide SEQ ID NO: 25, and oligonucleotide SEQ ID NO: 26.

24. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of claim 23.

25. The method according to claim 3, wherein said target DNA is a gene of Vibrio cholerae and said primers are selected from the group consisting of oligonucleotides of claims 23 and 24.

26. The method according to claim 25, wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 23 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 25; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 24 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 26.

27. The kit according to claim 4, wherein said pair of primers are selected from the group consisting of oligonucleotides of claims 23 and 24 for detection of Vibrio cholerae.

28. The synthetic oligonucleotide according to claim 1, wherein said gene is the enterotoxin gene of Clostridium perfringens, and wherein the synthetic oligonucleotide comprises at least 10 consecutive bases of the oligonucleotide selected from the group consisting of oligonucleotide SEQ ID NO: 27, oligonucleotide SEQ ID NO: 28, oligonucleotide SEQ ID NO: 29, oligonucleotide SEQ ID NO: 30, oligonucleotide SEQ ID NO: 31, oligonucleotide SEQ ID NO: 32, oligonucleotide SEQ ID NO: 33, oligonucleotide SEQ ID NO: 34, and oligonucleotide SEQ ID NO: 35.

29. The synthetic oligonucleotide according to claim 2, wherein said nucleotide sequence is complementary to the synthetic oligonucleotide of claim 28.

30. The method according to claim 3, wherein said target DNA is a gene of Clostridium perfringens and said primers are selected from the group consisting of oligonucleotides comprising at least 10 consecutive bases of the oligonucleotide of SEQ ID NO: 27, the oligonucleotide of SEQ ID NO: 28, the oligonucleotide of SEQ ID NO: 29, the oligonculeotide SEQ ID NO: 30, the oligonucleotide of SEQ ID NO: 31, the oligonucleotide of SEQ ID NO: 32, the oligonucleotide of SEQ ID NO: 33, the oligonucleotide of SEQ ID NO: 34, the oliogonucleotide of SEQ ID NO: 35, the oligonucleotide complementary to SEQ ID NO: 27, the oligonucleotide complementary to SEQ ID NO: 28, the oligonucleotide complementary to SEQ ID NO: 29, the oligonucleotide complementary to SEQ ID NO: 30 and the oligonucleotide complementary to SEQ ID NO: 31, the oligonucleotide complementary to SEQ ID NO: 32, the oligonucleotide complementary to SEQ ID NO: 33, the oligonucleotide complementary to SEQ ID NO: 34 and the oligonucleotide complementary to SEQ ID NO: 35.

31. The method according to claim 30, wherein said two primers are selected from the following oligonucleotide combinations: a combination in which one. oligonucleotide comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 27 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 32; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 28 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 33; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 29 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 33; a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 30 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 34; and a combination in which one comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 31 and the other comprises at least 10 consecutive bases of oligonucleotide SEQ ID NO: 35.

32. The kit according to claim 4, wherein said pair of primers are selected from the group consisting of oligonucleotides of claims 28 and 29 for detection of Clostridium perfringens.