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WO2013007996A1 - Dosage de détection de salmonella - Google Patents

Dosage de détection de salmonella Download PDF

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Publication number
WO2013007996A1
WO2013007996A1 PCT/GB2012/051614 GB2012051614W WO2013007996A1 WO 2013007996 A1 WO2013007996 A1 WO 2013007996A1 GB 2012051614 W GB2012051614 W GB 2012051614W WO 2013007996 A1 WO2013007996 A1 WO 2013007996A1
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WO
WIPO (PCT)
Prior art keywords
seq
probe
nucleotide
bind
complement
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PCT/GB2012/051614
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English (en)
Inventor
John Wain
Mark Achtman
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Health Protection Agency
University College Cork - National University Of Ireland
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Application filed by Health Protection Agency, University College Cork - National University Of Ireland filed Critical Health Protection Agency
Publication of WO2013007996A1 publication Critical patent/WO2013007996A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for the detection of Hazard Group 3 (HG3) Salmonella enterica serovar Typhi (S. Typhi), S. Paratyphi A, S. Paratyphi B, and S. Paratyphi C). Also provided are corresponding probes and primers.
  • HG3 Salmonella enterica serovar Typhi S. Typhi
  • S. Paratyphi A Salmonella enterica serovar Typhi
  • S. Paratyphi B S. Paratyphi B
  • S. Paratyphi C S. Paratyphi C
  • probes and primers also provided.
  • HG3 refers to a classification system introduced by the Advisory Committee on Dangerous Pathogens (http://www.dh.gov.uk/ab/ACDP/index.htm).
  • the Advisory Committee on Dangerous Pathogens (ACDP) is a UK-wide advisory non-departmental public body. It was established in 1981 , and the terms of reference were revised in 1991 and 201 1 to allow for a wider remit.
  • S. Typhi and S. Paratyphi are invasive, life-threatening human bacterial pathogens, causing the systemic diseases enteric fever which continues to pose significant threats for public health, especially in many developing countries.
  • a population based study estimated that the global disease burdens associated with typhoid and paratyphoid fever were 21.6 million cases (with 216,000 deaths) and 5.4 million cases, respectively - Crump, J.A. et al. (2004) Bull World Hith Org 82, pp. 346-53.
  • clinical samples are screened for the presence or absence of HG3 Salmonella enterica.
  • HG3 bacterium If a HG3 bacterium is detected the sample in question must be processed in a high-level containment laboratory, containment level 3 (CL-3) laboratory, under stringent, high-level containment procedures, and by laboratory personnel skilled in handling HG3 organisms. In contrast, if no HG3 salmonella bacterium is detected (for example, only HG2 salmonella bacteria such as Enteritidis, Typhimuruim, or Choleraesuis are detected), the sample in question may be processed in a low-level containment laboratory, with associated less stringent operating procedures, and without the need for highly skilled HG3 personnel.
  • CL-3 containment level 3
  • HG3 containment The costs involved with HG3 containment are significant, and considerably higher (in terms of laboratory equipment and consumables, increased processing time due to added exposure risk, and use of highly-skilled personnel) than are the corresponding costs associated with low-level containment.
  • HG3 salmonella detection systems typically rely on a combination of antiserum tests and biochemical tests, which are not only time-consuming (typically taking
  • HG3 detection systems are sub-optimal, and result in the generation of undesirable false-positives (i.e. salmonella assigned to HG3, when, in fact, they should have been assigned to a lower category such as HG2).
  • the present invention addresses one or more of the above-described problems.
  • a first aspect of the present invention provides a method of screening a sample containing nucleic acid for the presence or absence of HG3 salmonella nucleic acid, said method comprising:
  • probe A wherein probe A binds to:
  • probe A does not bind to the nucleotide at position 328 of SEQ ID NO: 2 when the nucleotide at position 328 is C; ii. probe B, wherein probe B binds to:
  • probe B does not bind to the nucleotide at position 136 of SEQ ID NO: 4 when the nucleotide at position 136 is G;
  • probe C wherein probe C binds to:
  • probe C does not bind to the nucleotide at position 378 of SEQ ID NO: 6 when the nucleotide at position 378 is A;
  • probe D wherein probe D binds to:
  • probe E binds to:
  • probe F wherein probe F binds to: i. position 69 of target SEQ ID NO: 1 1 when the nucleotide at position 69 of SEQ ID NO: 1 1 is A, and wherein probe F does not bind to position 69 of SEQ ID NO: 1 1 when the nucleotide at position 69 is G; or
  • probe F does not bind to the nucleotide at position 69 of SEQ I D NO: 1 1 when the nucleotide at position 69 is C;
  • probe G wherein probe G binds to:
  • G does not bind to the nucleotide at position 204 of SEQ ID NO:
  • probe H wherein probe H binds to:
  • probe I wherein probe I binds to:
  • probe I does not bind to the nucleotide at position 300 of SEQ ID NO: 18 when the nucleotide at position 300 is A;
  • probe J binds to: i. position 342 of target SEQ ID NO: 19 when the nucleotide at position 342 of SEQ ID NO: 19 is C, and wherein probe J does not bind to position 342 of SEQ ID NO: 19 when the nucleotide at position 342 is A or T; or
  • probe K wherein probe K binds to:
  • probe K does not bind to the nucleotide at position 252 of SEQ ID NO: 22 when the nucleotide at position 252 is C;
  • probe L wherein probe L binds to:
  • probe L does not bind to the nucleotide at position 9 of SEQ ID NO: 24 when the nucleotide at position 9 is A;
  • the method of present invention provides a highly accurate (e.g. greater than 99%) and/ or rapid (e.g. within 48, 36 or 24 hours) screening system for confirming the presence or absence of HG3 salmonella. This represents a significant improvement over existing HG3 salmonella detection systems.
  • all of the probes A-L are added substantially simultaneously and/ or contact the nucleic acid sample substantially simultaneously.
  • This multiplex embodiment has the advantage of minimising the overall time required to complete the method of the invention.
  • the method is performed in a cyclical manner, each cycle comprising the introduction of one or more of said probe(s) A-L.
  • each of said probes A-L may be added prior to, simultaneously with, or subsequent to one another.
  • the method may include a washing step (eg. between steps B and C) to remove unbound probe(s).
  • the detection step C may be performed on bound probe and/ or on unbound probe.
  • the detection step C may be performed on unbound probe.
  • the initially bound probe may be released (eg. by changing an assay parameter such as temperature and/ or pH), and the released (unbound) probe then detected.
  • the method may further comprise use of probe M, wherein probe M binds to:
  • probe M does not bind to the nucleotide at position 67 of SEQ ID NO: 26 when the nucleotide at position 67 is T, and wherein probe M does not bind to the nucleotide at position 67 of SEQ ID NO: 26 when the nucleotide at position 67 is C;
  • the binding of probe M to its target nucleic acid sequence (or the complement thereof) when in combination with the binding of any one of probes C-L to their respective target nucleic acid sequences (or the complement thereof), provides additional confirmation of the presence of HG3 salmonella nucleic acid.
  • the method of the present invention may further comprise confirming the presence or absence of HG2 salmonella nucleic acid.
  • Said HG2 detection method may be performed prior to, simultaneously with, or subsequent to any one of the HG3 detection method steps.
  • HG2 nucleic acid is confirmed by the binding (to sample nucleic acid) of one or more probe selected from the group consisting of probes N-U, wherein:
  • probe N does not bind to the nucleotide at position 385 of SEQ ID NO: 28 when the nucleotide at position 385 is G;
  • nucleotide at position 501 when the nucleotide at position 501 is G, and wherein probe O does not bind to the nucleotide at position 501 of SEQ ID NO: 30 when the nucleotide at position 501 is A;
  • probe P binds to
  • probe Q does not bind to the nucleotide at position 492 of SEQ ID NO: 34 when the nucleotide at position 492 is T;
  • probe R does not bind to the nucleotide at position 271 of SEQ ID NO: 36 when the nucleotide at position 271 is C;
  • probe S does not bind to the nucleotide at position 100 of SEQ ID NO: 38 when the nucleotide at position 100 is C;
  • probe T does not bind to the nucleotide at position 210 of SEQ ID NO: 40 when the nucleotide at position 210 is T;
  • probe U binds to
  • probe U does not bind to the nucleotide at position 66 of SEQ ID NO: 42 when the nucleotide at position 66 is G.
  • binding of one or more of probes A-B to sample nucleic acid confirms the presence of HG3 S. paratyphi A nucleic acid.
  • binding of one or more of probes C-L (or probe M when in combination with any of probes C-L) to sample nucleic acid confirms the presence of HG3 S. paratyphi B/ Java nucleic acid.
  • binding of one or more of probes J-K to sample nucleic acid confirms the presence of HG3 S. paratyphi C nucleic acid. In one embodiment, binding of probe L to sample nucleic acid confirms the presence of HG3 S. typhi nucleic acid.
  • binding of one or more of probes N-P to sample nucleic acid confirms the presence of HG2 S. enteritidis nucleic acid.
  • binding of one or more of probes Q-T to sample nucleic acid confirms the presence of HG2 S. typhimurium nucleic acid.
  • binding of probe U to sample nucleic acid confirms the presence of HG2 S. choleraesuis nucleic acid.
  • the present invention includes a method of 'serotyping' salmonella bacterial species. In more detail, the aforementioned methods may be performed to confirm (or deny) the presence of one or more specific types of salmonella selected from HG3 S. paratyphi A nucleic acid (via probe A and/ or probe B), HG3 S. paratyphi B/ Java (via one or more, such as all, of probes C-L), HG3 S. paratyphi C nucleic acid (via probe J and/ or probe K), HG3 S.
  • HG2 S. enteritidis nucleic acid via one or more, such as all, of probes N-P
  • HG2 S. typhimurium nucleic acid via one or more, such as all, of probes Q-T
  • HG2 S. choleraesuis via probe U.
  • probe A and/ or probe B may be used to serotype S. paratyphi A; one or more, such as all, of probes C-L may be used to serotype HG3 S. paratyphi B/ Java; probe J and/ or probe K may be used to serotype HG3 S. paratyphi C; probe L may be used to serotype HG3 S. typhi; one or more, such as all, of probes N-P may be used to serotype HG2 S. enteritidis; one or more, such as all, of probes Q-T may be used to serotype HG2 S.
  • probe U may be used to serotype HG2 S. choleraesuis.
  • the probes A-U of the present invention bind to a defined region on the target nucleic acid SEQ ID NOs (or complement thereof) as specified above, wherein said defined region includes the nucleic acid residue at the particular position as specified above.
  • the probe A binds to a region on SEQ ID NO: 1 that includes the nucleic acid residue A at position 328. However, the same probe A does not bind to the same region on SEQ ID NO: 1 when the nucleic acid residue at position 328 is G. Similarly, the probe A binds to a region on SEQ I D NO: 2 (ie. the complement of SEQ ID NO: 1 ) that includes the nucleic acid residue T at position 328. However, the same probe A does not bind to the same region on SEQ ID NO: 2 when the nucleic acid residue at position 328 is C.
  • probe B-U binds to a region on SEQ ID NO: 3 that includes the nucleic acid residue T at position 136.
  • the same probe B does not bind to the same region on SEQ ID NO: 3 when the nucleic acid residue at position 136 is C.
  • the probe B binds to a region on SEQ ID NO: 4 (ie. the complement of SEQ ID NO: 3) that includes the nucleic acid residue A at position 136.
  • the same probe B does not bind to the same region on SEQ ID NO: 4 when the nucleic acid residue at position 136 is G.
  • Each of probes A-U typically comprises a sequence of 9-30 (or longer) consecutive nucleotides. It is this sequence that binds the probe to the defined region on the respective target nucleic acid SEQ ID NO (or complement thereof) as specified above.
  • the probes may contain additional nucleotides, though said additional nucleotides need not necessarily contribute to the binding of the probe to its target nucleic acid (or complement thereof). In this regard, additional nucleotides may be included to help provide stability to the probe. Similarly, additional nucleotides may be added (typically to the 5' end) to provide a labelling means for downstream detection.
  • each of probes A-U may comprise a sequence of at least 9, at least 12, at least 15, at least 18, at least 21 , at least 24, or at least 27 consecutive nucleotides that bind the probe to its target nucleic acid (or complement thereof).
  • each of the probes A-U may comprise at most 30, at most 27, at most 24, at most 21 , at most 18, at most 15, or at most 12 consecutive nucleotides that bind the probe to its target nucleic acid (or complement thereof).
  • Each of probes A-U binds to a defined region on its respective target nucleic acid SEQ ID NO (or complement thereof) as specified above. In this regard, it is self-explanatory that the length of the defined region typically matches the length of the probe.
  • said probe will typically bind to a corresponding defined region of 20 consecutive nucleotides on the target sequence (or complement thereof).
  • Each of probes A-U binds to its respective target nucleic acid SEQ ID NO (or complement thereof) as specified above. In order to do so, each of said probes is complementary in sequence to its respective target nucleic acid SEQ ID NO (or complement thereof).
  • each of probes A-U includes a key nucleotide that forms a natural base-pairing with the particular nucleotide (at the particular position) as specified above for each target nucleic acid SEQ ID NO (or complement thereof).
  • Reference to a natural base-pairing means that, when the specified nucleotide on the target SEQ ID NO (or complement thereof) is A, then the probe contains a key nucleotide T that binds the probe to the A nucleotide present on the target SEQ ID NO (or complement thereof). Similarly, when the specified nucleotide on the target SEQ ID NO (or complement thereof) is T, then the probe will contain a key nucleotide A that binds the probe to the T nucleotide present on the target SEQ ID NO (or complement thereof).
  • the probe when the specified nucleotide on the target SEQ ID NO (or complement thereof) is G, then the probe will contain a key nucleotide C that binds the probe to the G nucleotide present on the target SEQ ID NO (or complement thereof). Similarly, when the specified nucleotide on the target SEQ ID NO (or complement thereof) is C, then the probe will contain a key nucleotide G that binds the probe to the C nucleotide present on the target SEQ ID NO (or complement thereof).
  • Probes A-U have a 5'-to-3' orientation.
  • the key nucleotide may be located at any position within the probe.
  • the key nucleotide may be located within 5 nucleotides of the 3' end of the probe.
  • the key nucleotide may be located at the 3' end (ie. the extreme 3' nucleotide position) of the probe.
  • the key nucleotide may be located 1 , 2, 3, or 4 nucleotides in (ie. in a 5' direction) from the extreme 3' nucleotide position of the probe.
  • each of the probes A-U comprises (or consists of) a nucleotide sequence having at least 80% identity to (eg.
  • nucleotide sequence selected from SEQ ID NOs: 395-436 (as shown in Table 1 ), though with the proviso that the key nucleotide (underlined) is not changed. Conservative substitutions are preferred.
  • the present invention also embraces shorter probe sequences thereof (as described above), though with the proviso that the key nucleotide (underlined) is not changed.
  • each of the probes A-U comprises (or consists of) a nucleotide sequence as shown in Table 1 , though differing at no more than 5 nucleotide positions (for example at no more than 4, 3, 2 or 1 nucleotide position), though again with the proviso that the key nucleotide (underlined) is not changed. Conservative substitutions are preferred.
  • the present invention also embraces shorter probe sequences thereof (as described above), though with the proviso that the key nucleotide (underlined) is not changed.
  • the probes of the invention bind to critical nucleotides (present at specific positions) on genes of HG3 (and/ or HG2) salmonella.
  • probe A binds to 328A of SEQ ID NO: 1 , but not when the nucleotide at position 328 is G.
  • SEQ ID NO: 1 corresponds to a S. paratyphi A aroC gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 43-52.
  • probe A also binds to 328A of any of SEQ ID NOs: 43-52, but not when the nucleotide at position 328 is G.
  • probe A also binds to 328T of any of SEQ ID NOs: 53- 62, but not when the nucleotide at position 328 is C.
  • Probe B binds to 136T of SEQ ID NO: 3, but not when the nucleotide at position 136 is C.
  • SEQ ID NO: 3 corresponds to a S. paratyphi A thr A gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 63-72.
  • probe B also binds to 136T of any of SEQ ID NOs: 63-72, but not when the nucleotide at position 136 is C.
  • SEQ ID NO: 3 ie. SEQ ID NO: 4
  • SEQ ID NOs: 73-82 represent strain variants of SEQ ID NO: 4.
  • probe B also binds to 136A of any of SEQ ID NOs: 73-82, but not when the nucleotide at position 136 is G.
  • Probe C binds to 378C of SEQ ID NO: 5, but not when the nucleotide at position 378 is T.
  • SEQ ID NO: 5 corresponds to a S. paratyphi B/ java dnaN gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 83-92.
  • probe C also binds to 378C of any of SEQ ID NOs: 83-92, but not when the nucleotide at position 378 is T.
  • SEQ ID NO: 6 ie. SEQ ID NO: 5
  • SEQ ID NOs: 93-102 represent strain variants of SEQ ID NO: 5.
  • probe C also binds to 378G of any of SEQ ID NOs: 93-102, but not when the nucleotide at position 378 is A.
  • Probe D binds to 57C of SEQ ID NO: 7, but not when the nucleotide at position 57 is T.
  • SEQ ID NO: 7 corresponds to a S. paratyphi B/ java dnaN gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 103- 1 12.
  • probe D also binds to 57C of any of SEQ ID NOs: 103-1 12, but not when the nucleotide at position 57 is T.
  • SEQ ID NO: 7 ie. SEQ ID NO: 8
  • SEQ ID NOs: 1 13-122 represent strain variants of SEQ ID NO: 7.
  • probe D also binds to 57G of any of SEQ ID NOs: 1 13-122, but not when the nucleotide at position 57 is A.
  • Probe E binds to 339C of SEQ ID NO: 9, but not when the nucleotide at position 339 is T.
  • SEQ ID NO: 9 corresponds to a S. paratyphi B/ java hisD gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 123- 132.
  • probe E also binds to 339C of any of SEQ ID NOs: 123-132, but not when the nucleotide at position 339 is T.
  • SEQ ID NO: 9 ie. SEQ ID NO: 10
  • SEQ ID NOs: 133-142 represent strain variants of SEQ ID NO: 10.
  • probe E also binds to 339G of any of SEQ ID NOs: 133-142, but not when the nucleotide at position 339 is A.
  • Probe F binds to 69A of SEQ ID NO: 1 1 , but not when the nucleotide at position 69 is G.
  • SEQ ID NO: 1 1 corresponds to a S. paratyphi B/ java hisD gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 143- 152.
  • probe F also binds to 60A of any of SEQ ID NOs: 143-152, but not when the nucleotide at position 69 is G.
  • SEQ ID NO: 1 1 ie. SEQ ID NO: 12
  • SEQ ID NOs: 153-162 represent strain variants of SEQ ID NO: 12.
  • probe F also binds to 69T of any of SEQ ID NOs: 153-162, but not when the nucleotide at position 69 is C.
  • Probe G binds to 204G of SEQ ID NO: 13, but not when the nucleotide at position 204 is A.
  • SEQ ID NO: 13 corresponds to a S. paratyphi B/ java hisD gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 163- 172.
  • probe G also binds to 204G of any of SEQ ID NOs: 163-172, but not when the nucleotide at position 204 is A.
  • SEQ ID NO: 14 the complement of SEQ ID NO: 13 (ie. SEQ ID NO: 14) in that SEQ ID NOs: 173-182 represent strain variants of SEQ ID NO: 14.
  • probe G also binds to 204C of any of SEQ ID NOs: 173-182, but not when the nucleotide at position 204 is T.
  • Probe H binds to 255A of SEQ I D NO: 15, but not when the nucleotide at position 255 is G.
  • SEQ ID NO: 15 corresponds to a S. paratyphi B/ java sucA gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 183- 192.
  • probe H also binds to 255A of any of SEQ I D NOs: 183-192, but not when the nucleotide at position 255 is G.
  • SEQ ID NO: 15 ie. SEQ ID NO: 16
  • SEQ ID NOs: 193-202 represent strain variants of SEQ ID NO: 16.
  • probe H also binds to 255T of any of SEQ ID NOs: 193-202, but not when the nucleotide at position 255 is C.
  • Probe I binds to 300C of SEQ ID NO: 17, but not when the nucleotide at position 300 is T.
  • SEQ ID NO: 17 corresponds to a S. paratyphi B/ java sucA gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 203- 212.
  • probe I also binds to 300C of any of SEQ ID NOs: 203-212, but not when the nucleotide at position 300 is T.
  • SEQ ID NO: 18 the complement of SEQ ID NO: 17 (ie. SEQ ID NO: 18) in that SEQ ID NOs: 213-222 represent strain variants of SEQ ID NO: 18.
  • probe I also binds to 300G of any of SEQ ID NOs: 213-222, but not when the nucleotide at position 300 is A.
  • Probe J binds to 342C of SEQ ID NO: 19, but not when the nucleotide at position 342 is A or T.
  • SEQ ID NO: 19 corresponds to a S. paratyphi C dnaN gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 223- 232.
  • probe J also binds to 342C of any of SEQ ID NOs: 223-232, but not when the nucleotide at position 342 is A or T.
  • SEQ ID NO: 19 ie. SEQ ID NO: 20
  • SEQ ID NOs: 233-242 represent strain variants of SEQ I D NO: 20.
  • probe J also binds to 342G of any of SEQ ID NOs: 233-242, but not when the nucleotide at position 342 is T or A.
  • Probe K binds to 252T of SEQ ID NO: 21 , but not when the nucleotide at position 252 is G.
  • SEQ ID NO: 21 corresponds to a S. paratyphi C sucA gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 243- 252.
  • probe K also binds to 352T of any of SEQ ID NOs: 243-252, but not when the nucleotide at position 252 is G.
  • SEQ ID NO: 21 ie. SEQ ID NO: 22
  • SEQ ID NOs: 253-262 represent strain variants of SEQ ID NO: 22.
  • probe K also binds to 252A of any of SEQ ID NOs: 253-262, but not when the nucleotide at position 252 is C.
  • Probe L binds to 9C of SEQ ID NO: 23, but not when the nucleotide at position 9 is T.
  • SEQ ID NO: 23 corresponds to a S. paratyphi C aroC gene sequence, and strain variants of said sequence are known in the art.
  • probe L also binds to 9C of any of said strain variant SEQ ID NOs, but not when the nucleotide at position 9 is T
  • SEQ ID NO: 24 the complement of SEQ ID NO: 23 (ie. SEQ ID NO: 24) in that strain variants of SEQ ID NO: 24 are known in the art.
  • probe L also binds to 9G of any of said variant SEQ ID NOs, but not when the nucleotide at position 9 is A.
  • Probe M binds to 67A of SEQ ID NO: 25, but not when the nucleotide at position 67 is G.
  • SEQ ID NO: 25 corresponds to a S. paratyphi B tartrate gene sequence, and strain variants of said sequence are illustrated, for example, in SEQ ID NO: 263.
  • probe M also binds to 67A of any of said variant SEQ ID NOs, but not when the nucleotide at position 67 is G.
  • probe M also binds to 67T of any of strain variant SEQ ID NOs, but not when the nucleotide at position 67 is C.
  • Probe N binds to 385T of SEQ ID NO: 27, but not when the nucleotide at position 385 is C.
  • SEQ ID NO: 27 corresponds to a S. enteritidis hisD gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 265- 267.
  • probe N also binds to 385T of any of SEQ ID NOs: 265-267, but not when the nucleotide at position 385 is C.
  • SEQ ID NO: 28 the complement of SEQ ID NO: 27 (ie. SEQ ID NO: 28) in that SEQ ID NOs: 268-270 represent strain variants of SEQ ID NO: 28.
  • probe N also binds to 385A of any of SEQ ID NOs: 268-270, but not when the nucleotide at position 385 is G.
  • Probe O binds to 501 C of SEQ ID NO: 29, but not when the nucleotide at position 501 is T.
  • SEQ ID NO: 29 corresponds to a S. enteritidis hisD gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 271 - 280.
  • probe O also binds to 501 C of any of SEQ ID NOs: 271 -280, but not when the nucleotide at position 501 is T.
  • the same applies to the complement of SEQ ID NO: 29 ie.
  • SEQ ID NO: 30 in that SEQ ID NOs: 281 -290 represent strain variants of SEQ ID NO: 30.
  • probe O also binds to 501 G of any of SEQ ID NOs: 281 -290, but not when the nucleotide at position 501 is A.
  • Probe P binds to 271 G of SEQ I D NO: 31 , but not when the nucleotide at position 271 is T.
  • SEQ ID NO: 31 corresponds to a S. enteritidis purE gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 291 - 300.
  • probe P also binds to 271 G of any of SEQ ID NOs: 291 -300, but not when the nucleotide at position 271 is T.
  • SEQ ID NO: 32 the complement of SEQ ID NO: 31 (ie. SEQ ID NO: 32) in that SEQ ID NOs: 301 -310 represent strain variants of SEQ ID NO: 32.
  • probe P also binds to 271 C of any of SEQ ID NOs: 301 -310, but not when the nucleotide at position 271 is A.
  • Probe Q binds to 492G of SEQ ID NO: 33, but not when the nucleotide at position 492 is A.
  • SEQ ID NO: 33 corresponds to a S. typhimurium aroC gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 31 1 - 320.
  • probe Q also binds to 492G of any of SEQ ID NOs: 31 1 -320, but not when the nucleotide at position 492 is A.
  • the same applies to the complement of SEQ ID NO: 33 ie.
  • SEQ ID NO: 34 in that SEQ ID NOs: 321 -330 represent strain variants of SEQ ID NO: 34.
  • probe Q also binds to 492C of any of SEQ ID NOs: 321 -330, but not when the nucleotide at position 492 is T.
  • Probe R binds to 271 T of SEQ ID NO: 35, but not when the nucleotide at position 271 is G.
  • SEQ ID NO: 35 corresponds to a S. typhimurium dnaN gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 331 - 338.
  • probe R also binds to 271 T of any of SEQ ID NOs: 331 -338, but not when the nucleotide at position 271 is G.
  • SEQ ID NO: 35 ie. SEQ ID NO: 36
  • SEQ ID NOs: 339-346 represent strain variants of SEQ ID NO: 36.
  • probe R also binds to 271A of any of SEQ ID NOs: 339-346, but not when the nucleotide at position 271 is C.
  • Probe S binds to 100T of SEQ ID NO: 37, but not when the nucleotide at position 100 is G.
  • SEQ ID NO: 37 corresponds to a S. typhimurium hemD gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 347- 356.
  • probe S also binds to 100T of any of SEQ ID NOs: 347-356, but not when the nucleotide at position 100 is G.
  • SEQ ID NO: 38 the complement of SEQ ID NO: 37 (ie. SEQ ID NO: 38) in that SEQ ID NOs: 357-366 represent strain variants of SEQ ID NO: 38.
  • probe S also binds to 100A of any of SEQ ID NOs: 357-366, but not when the nucleotide at position 100 is C.
  • Probe T binds to 210T of SEQ ID NO: 39, but not when the nucleotide at position 210 is A.
  • SEQ ID NO: 39 corresponds to a S. typhimurium thrA gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 367- 376.
  • probe T also binds to 210T of any of SEQ ID NOs: 367-376, but not when the nucleotide at position 201 is A.
  • SEQ ID NO: 40 the complement of SEQ ID NO: 39 (ie. SEQ ID NO: 40) in that SEQ ID NOs: 377-386 represent strain variants of SEQ ID NO: 40.
  • probe T also binds to 210A of any of SEQ ID NOs: 377-386, but not when the nucleotide at position 210 is T.
  • Probe U binds to 66T of SEQ ID NO: 41 , but not when the nucleotide at position 66 is C.
  • SEQ ID NO: 41 corresponds to a S. cholera aroC gene sequence, and strain variants of said sequence are illustrated in SEQ ID NOs: 387-390.
  • probe U also binds to 66T of any of SEQ ID NOs: 387-390, but not when the nucleotide at position 66 is C.
  • probe U also binds to 66A of any of SEQ ID NOs: 391 -394, but not when the nucleotide at position 66 is G.
  • the probes of the invention are designed to hybridise to their target (or complement) sequence. It is preferred that the binding conditions are such that a high level of specificity is provided - ie. hybridisation of the probe occurs under "stringent conditions". In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target (or complement) sequence hybridises to a perfectly matched probe.
  • the T m of probes of the present invention at a salt concentration of about 0.02M or less at pH 7, is for example above 60°C, such as about 70°C.
  • Premixed binding solutions are commercially available (eg. EXPRESSHYB Hybridisation Solution from CLONTECH Laboratories, Inc.), and hybridisation can be performed according to the manufacturer's instructions.
  • Preferred probes of the present invention are selected so as to have minimal homology with human DNA.
  • the selection process may involve comparing a candidate probe sequence with human DNA and rejecting the probe if the homology is greater than 50%.
  • the aim of this selection process is to reduce annealing of probe to contaminating human DNA sequences and hence allow improved specificity of the assay.
  • any of the probes described herein may comprise a tag and/ or label.
  • the tag and/ or label may, for example, be located (independently of one another) towards the middle or towards or at the 5' or 3' end of the herein described probes, for example at the 5' end.
  • the tag/ label is associated with the HG3 target nucleic acid (or complement thereof).
  • the probes may act as primers during the method of the invention and the tag/ label may therefore become incorporated into the amplification product as the primer is extended.
  • suitable labels include detectable labels such as radiolabels or fluorescent or coloured molecules, enzymatic markers or chromogenic markers - eg. dyes that produce a visible colour change upon hybridisation of the probe.
  • the label may be digoxygenin, fluorescein-isothiocyanate (FITC), R-phycoerythrin, Alexa 532 or Cy3.
  • the label may be a reporter molecule, which is detected directly, such as by exposure to photographic or X-ray film.
  • the label is not directly detectable, but may be detected indirectly, for example, in a two-phase system.
  • An example of indirect label detection is binding of an antibody to the label.
  • tags include "complement/ anti-complement pairs".
  • the term "complement/ anti-complement pair” denotes non-identical moieties that form a non- covalently associated, stable pair under appropriate conditions.
  • suitable tags include biotin and streptavidin (or avidin).
  • a biotin tag may be captured using streptavidin, which may be coated onto a substrate or support such as a bead (for example a magnetic bead) or membrane.
  • a streptavidin tag may be captured using biotin, which may be coated onto a substrate or support such as a bead (for example a magnetic bead) or membrane.
  • complement/ anti-complement pairs include receptor/ ligand pairs, antibody/ antigen (or hapten or epitope) pairs, and the like.
  • Another example is a nucleic acid sequence tag that binds to a complementary sequence. The latter may itself be pre-labelled, or may be attached to a surface (eg. a bead) which is separately labelled.
  • An example of the latter embodiment is the well-known Luminex R bead system.
  • Other exemplary pairs of tags and capture molecules include receptor/ ligand pairs and antibody/ antigen (or hapten or epitope) pairs. Where subsequent dissociation of the complement/ anti- complement pair is desirable, the complement/ anti-complement pair has a binding affinity of, for example, less than 10 9 M "1 .
  • probes A-U may be labelled with different labels or tags, thereby allowing separate identification of each probe when used in the method of the present invention.
  • one or more of probes A-U may be labelled with different nucleic acid sequences (aka 'sequence tags' or 'sequence labels').
  • Said different nucleic acid sequence labels may be attached, for example, to the 5' end of the defined sequence of the probe that binds to the target nucleic acid SEQ ID NO (or complement thereof), thereby allowing separate identification of each probe when used in the method of the present invention.
  • each of said nucleic acid tags is able to bind to a different, complementary nucleic acid sequence present on a surface (eg.
  • nucleic acid tags are illustrated in Table 2.
  • nucleic acid tags Any conventional method may be employed to attach nucleic acid tags to a probe of the present invention (eg. to the 5' end of the defined binding region of the probe).
  • nucleic acid probes of the invention may be constructed by commercial providers. Examples of probes of the invention to which tags illustrated in Table 2 have been attached are illustrated in Table 3.
  • the sample is for example a clinical sample (or is derived from a clinical sample) such as: faeces or blood, sputum, bronchoalveolar lavage, tracheal aspirate, lung tissue samples, cerebrospinal fluid, archaeological samples.
  • a clinical sample or is derived from a clinical sample
  • faeces or blood such as: faeces or blood, sputum, bronchoalveolar lavage, tracheal aspirate, lung tissue samples, cerebrospinal fluid, archaeological samples.
  • an amplification step may be introduced.
  • amplification may be carried out using methods and platforms known in the art, for example PCR, such as real-time PCR, block-based PCR, ligase chain reaction, glass capillaries, isothermal amplification methods including loop-mediated isothermal amplification, rolling circle amplification transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification.
  • PCR such as real-time PCR, block-based PCR, ligase chain reaction, glass capillaries
  • isothermal amplification methods including loop-mediated isothermal amplification, rolling circle amplification transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, isothermal multiple displacement amplification,
  • amplification can be carried using any amplification platform - as such, an advantage of this embodiment of the assay is that it is platform independent and not tied to any particular instrument.
  • a general amplification step eg. pre-detection
  • PCR amplification primers are typically employed to amplify approximately 100-400 base pair regions of the target/ complementary nucleic acid that contain the HG3 (and/ or HG2) nucleotide targets of the present invention.
  • forward and reverse primers are extended in a 5' to 3' direction, thereby initiating the synthesis of new nucleic acid strands that are complementary to the individual strands of the target/ complementary HG3 nucleic acid.
  • the primers thereby drive amplification of HG3 (and/ or HG2) nucleic acid sequences, thereby generating amplification products comprising said HG3 (and/ or HG2) nucleic acid sequences.
  • an amplification step may be employed in which the probes of the present invention act as primers.
  • the probes act as primers
  • the probes are extended from their 3' ends (ie. in a 5'-to-'3') direction.
  • the resulting amplification products typically comprise 100-400 base pair regions of the target/ complementary nucleic acid.
  • This embodiment may be employed in conjunction with a general amplification step, such as the one described above.
  • the detection step may be carried out by any known means.
  • the probe or amplification product may be tagged and/ or labelled, and the detection method may therefore comprise detecting said tag and/ or label.
  • the probe(s) may comprise a tag and/ or label.
  • the tag/ label becomes associated with the target (or complement) nucleic acid.
  • the assay may comprise detecting the tag/ label and correlating presence of tag/ label with presence of HG3.
  • tag and/ or label may be incorporated during extension of the probe(s).
  • the amplification product(s) become tagged/ labelled, and the assay may therefore comprise detecting the tag/ label and correlating presence of tag/ label with presence of amplification product, and hence the presence of HG3 (and/ or HG2).
  • the amplification product may incorporate a tag/ label (eg. via a tagged/ labelled dNTP such as biotin-dNTP) as part of the amplification process, and the assay may further comprise the use of a binding partner complementary to said tag (eg. streptavidin) that includes a detectable tag/ label (eg. a fluorescent label, such as R-phycoerythrin).
  • a detectable tag/ label eg. a fluorescent label, such as R-phycoerythrin
  • the amplified product incorporates a detectable tag/ label (eg. a fluorescent label, such as R-phycoerythrin).
  • the probe(s) and/ or the amplification product(s) may include a further tag/ label (as the complement component) to allow capture of the amplification product(s).
  • a “complement/ anti-complement” pairing may be employed in which an anti-complement capture component binds to said further tag/ label (complement component) and thereby permits capture of the probe(s) and/ or amplification product(s).
  • suitable "complement/ anti-complement” partners have been described earlier in this specification, such as a complementary pair of nucleic acid sequences, a complementary antibody-antigen pair, etc.
  • the anti-complement capture component may be attached (eg. coated) on to a substrate or solid support - examples of suitable substrates/ supports include membranes and/ or beads (eg. a magnetic or fluorescent bead). Capture methods are well known in the art. For example, Luminex R beads may be employed. Alternatively, the use of magnetic beads may be advantageous because the beads (plus captured, tagged/ labelled amplification product) can easily be concentrated and separated from the sample, using conventional techniques known in the art.
  • Immobilisation provides a physical location for the anti-complement capture component (or probes), and may serve to fix the capture component/ probe at a desired location and/ or facilitate recovery or separation of probe.
  • the support may be a rigid solid support made from, for example, glass or plastic, such as a bead (for example a fluorescent or magnetic bead).
  • the support may be a membrane, such as nylon or nitrocellulose membrane.
  • 3D matrices are also suitable supports for use with the present invention - eg. polyacrylamide or PEG gels.
  • Immobilisation to a support/ platform may be achieved by a variety of conventional means.
  • immobilisation onto a support such as a nylon membrane may be achieved by UV cross-linking.
  • biotin-labelled molecules may be bound to streptavidin-coated substrates (and vice-versa), and molecules prepared with amino linkers may be immobilised on to silanised surfaces.
  • Another means of immobilisation is via a poly-T tail or a poly-C tail, for example at the 3' or 5' end.
  • probes A-L (optionally also probe M; optionally also probes N-U with or without probe M) of the invention comprise a nucleic acid sequence tag/ label (eg. attached to each probe at the 5' end of the defined sequence of the probe that binds to target/ complement nucleic acid).
  • each of the probes is provided with a different nucleic acid sequence tag/ label, wherein each of said tags/ labels (specifically) binds to a complementary nucleic acid sequence present on the surface of a bead.
  • Each of the different tags/ labels binds to its complementary sequence counterpart (and not to any of the complementary sequence counterparts of the other tags), which is located on a uniquely identifiable bead.
  • the beads are uniquely identifiable, for example by means of fluorescence at a specific wavelength.
  • probes of the invention bind to target/ complement nucleic acid (if present in the sample). Thereafter, (only) the bound probes may be extended (in the 3' direction) in the presence of one or more labelled dNTP (eg. biotin labelled dNTPs, such as biotin- dCTPs).
  • the extended primers may be contacted with a binding partner counterpart to the labelled dNTPs (eg. a streptavidin labelled flurophore, such as streptavidin labelled R- phycoerythrin), which binds to those labelled dNTPs that have become incorporated into the extended primers.
  • a binding partner counterpart to the labelled dNTPs eg. a streptavidin labelled flurophore, such as streptavidin labelled R- phycoerythrin
  • the labelled extended primers may be identified by allowing them to bind to their nucleic acid counterparts present on the uniquely identifiable beads. The latter may then be "called” (eg. to determine the type of bead present by wavelength emission) and the nature of the primer extension (and thus the type of target/ complement nucleic acid present) may be determined.
  • a simple schematic of one such embodiment is illustrated in Figure 1 .
  • HG3 detection probes For simplicity only two HG3 detection probes are illustrated - for illustrative purposes only, the first probe has a 3' nucleotide residue designated G and the second probe has a 3' nucleotide residue designated A. Each of the probes has a different 5' nucleic acid sequence tag.
  • Figure 1 illustrates a scenario in which only the second probe binds to salmonella nucleic acid present in the test sample.
  • the first probe does not bind to any nucleic acid present in the test sample.
  • the bound second probe is then extended in a 3' direction, and fluorescent label is incorporated as the probe becomes extended.
  • the present invention (a method of serotyping by PCR used for the detection of Hazard Group 3 (HG3) Salmonella) has been tested for use in the Laboratory for Gastrointestinal Pathogens at the HPA Colindale. Specific examples will now be given of the use of the invention in that laboratory. These are exemplary; they are not limiting in any way to the scope of the claims or to the invention. These examples represent the best practice for the using the invention as currently envisaged.
  • Example 1 The invention was tested for an ability to screen all Salmonella to allow release of HG2 Salmonella from the high level containment laboratory (CL). HG3 Salmonella must remain in the CL.
  • Salmonella Typhi (HG3): 150 tested and 150 correctly identified by the invention.
  • Salmonella Paratyphi A (HG3) 76 tested and 76 correctly identified by the invention;
  • Salmonella Paratyphi C (HG3) 34 tested and 34 correctly identified by the invention; of 260 HG3 isolates all were correctly identified.
  • Salmonella Enteritidis 25 tested and 25 correctly identified by the invention (1 not assigned as either HG3 or HG2).
  • the invention has an accuracy of 100% for the positive identification of 260 HG3 Salmonella and an accuracy of 106/106 for the positive identification of HG2 Salmonella.
  • the serious error rate (identification HG3 as HG2 Salmonella) was zero.
  • Example 2 The invention was tested using 356 unknown isolates as they were received by the Laboratory for Gastrointestinal Pathogens.
  • Salmonella Typhi (HG3): 17 tested and 17 correctly identified by the invention.
  • Salmonella Paratyphi A (HG3) 5 tested and 5 correctly identified by the invention; of 22 HG3 isolates all were correctly identified.
  • Other HG2 Salmonella 221 tested and 221 correctly identified by the invention; of 334 HG2 Salmonella tested 333 were correctly identified.
  • the invention when the operator is blind to the expected result of the test, has an accuracy of 22/22 for the positive identification of HG3 Salmonella and an accuracy of 333/334 for the positive identification of HG2 Salmonella.
  • the serious error rate (identification HG3 as HG2 Salmonella) was zero.

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Abstract

La présente invention concerne un procédé pour la détection de Salmonella enterica de sérovar Typhi (S. Typhi) de groupe de risque 3 (HG3), S. Paratyphi A, S. Paratyphi B, et S. Paratyphi C. L'invention concerne également des sondes et amorces correspondantes.
PCT/GB2012/051614 2011-07-08 2012-07-09 Dosage de détection de salmonella WO2013007996A1 (fr)

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CN105004857A (zh) * 2015-08-19 2015-10-28 上海市长宁区疾病预防控制中心 乙型副伤寒和爪哇变种沙门菌试剂盒、制备及使用方法
CN109468394A (zh) * 2018-11-30 2019-03-15 舟山出入境检验检疫局综合技术服务中心 一种检测四种沙门氏菌血清型的五重pcr引物、试剂盒及其应用
GB2612409A (en) * 2021-08-09 2023-05-03 Univ Jiangsu Indel molecular marker of ultrasonic mutagenesis salmonella typhimurium HISD gene and use thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105004857A (zh) * 2015-08-19 2015-10-28 上海市长宁区疾病预防控制中心 乙型副伤寒和爪哇变种沙门菌试剂盒、制备及使用方法
CN109468394A (zh) * 2018-11-30 2019-03-15 舟山出入境检验检疫局综合技术服务中心 一种检测四种沙门氏菌血清型的五重pcr引物、试剂盒及其应用
GB2612409A (en) * 2021-08-09 2023-05-03 Univ Jiangsu Indel molecular marker of ultrasonic mutagenesis salmonella typhimurium HISD gene and use thereof
EP4155414A4 (fr) * 2021-08-09 2023-12-20 Jiangsu University Marqueur moléculaire indel de gène hisd de salmonella typhimurium de mutagenèse ultrasonore et son utilisation
GB2612409B (en) * 2021-08-09 2024-08-21 Univ Jiangsu Indel molecular marker of ultrasonic mutagenesis salmonella typhimurium hisD gene and use thereof

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