# US 2014/0154692 A1 · Diagnostic methods for detecting clostridium difficile

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- Number: US 2014/0154692 A1
- Document ID: US20140154692A1
- Status: Published application
- Kind: A1
- Application number: 14/128,180
- Filed: 2012-06-25
- Published: 2014-06-05
- Earliest claimed: 2011-06-23
- Assignees: University of Ulster
- Inventors: Nigel G. Ternan; Geoffrey Mcmullan; Christopher I. Gill; Shailesh Jain
- Web page: https://patentgrep.com/patent/US20140154692A1

## CPC

- C12Q 1/689: for bacteria
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention provides a method of detecting Clostridium difficile in a sample, comprising detecting the presence in said sample of one or more genes that have been identified as being specific to Clostridium difficile. Also provided is a method of diagnosing a Clostridium difficile infection in a subject, a method of determining the efficacy of a therapeutic regime being used to treat a Clostridium difficile infection and a method of testing for the presence of Clostridium difficile in a sample. Further provided are primer pairs and a kit suitable for use in such methods.

## Claims

### Claim 1

A method of testing for the presence of Clostridium difficile in a sample, said method comprising analysing said sample for the presence of one or more genes selected from the group consisting, of CD3609, CD3617, CD3618, CD3635, CD3638, CD0638, CD1424, CD1487, CD1543a, CD1794, CD1906, CD2046, CD2098, CD2216, CD2264, CD2274, CD2309, CD3188, CD3288, CD3367 and CD2961 or a product of said genes.

### Claim 2

A method of detecting Clostridium difficile in a sample, said method comprising detecting the presence in said sample of one or more genes selected from the group consisting of CD3609, CD3617, CD3618, CD3635, CD3638, CD0638, CD1424, CD1487, CD1543a, CD1794, CD1906, CD2046, CD2098, CD2216, CD2264, CD2274, CD2309, CD3188, CD3288, CD3367 and CD2961, or a product of said genes.

### Claim 3 (depends on 1)

A method of diagnosing a Clostridium difficile infection in a subject, said method comprising performing the method of claim 1, wherein the sample has been obtained from said subject and the presence of one or more genes selected from the group consisting of CD3609, CD3617, CD3618, CD3635, CD3638, CD0638, CD1424, CD1487, CD1543a, CD1794, CD1906, CD2046, CD2098, CD2216, CD2264, CD2274, CD2309, CD3188, CD3288, CD3367 and CD2961, or a product of said genes, in the sample is diagnostic of a Clostridium difficile infection in said subject.

### Claim 4 (depends on 1)

A method of determining the efficacy of a therapeutic regime being used to treat a Clostridium difficile infection, said method comprising
(i) performing the method of claim 1 on a sample that has been obtained from a subject being treated for a Clostridium difficile infection; and
(ii) repeating step (i) on one or more further samples that have been obtained from the subject being treated for a Clostridium difficile infection.

### Claim 5

A primer pair selected from the group consisting of
(a) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 67 or nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 67; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 68 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 68;
(b) an isolated oligonucleotide comprising the nucleotide sequence of SEQ NO: 13 or a nucleotide sequence capable of hybridising, under high stringency conditions to the sequence complementary to SEQ ID NO: 13; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO 14 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 14;
(c) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO 15 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 15; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 16;
(d) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 17; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 18;
(e) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO:19 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO:19; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO:20;
(f) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO:37 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 37; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO:38;
(g) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 39 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 39; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 40 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 40;
(h) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 41 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 41; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 42 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 42;
(i) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 43 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 43; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO; 41 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 44;
(j) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 45 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 45; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 46 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 46;
(k) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 47 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 47; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 48 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 48;
(l) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 49 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 49; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO:50 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 50;
(m) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 51; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 52 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 52;
(n) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 53; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 54 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 54;
(o) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 55; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 56 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 56;
(p) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 57 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 57; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 58 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 58;
(q) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 59 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 59; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 60 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 60;
(r) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 61 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 61; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 62 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 62;
(s) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 63; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 64 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 64;
(t) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 65 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 65; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 66 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 66; and
(u) an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO: 11; and
an isolated oligonucleotide comprising the nucleotide sequence of SEQ ID NO 12 or a nucleotide sequence capable of hybridising under high stringency conditions to the sequence complementary to SEQ ID NO 12.

### Claim 6

A kit comprising one or more detection moieties for the detection of one or more genes selected from the group consisting of CD3609, CD3617, CD3618, CD3635, CD3638, CD0638, CD1424, CD1487, CD1543a, CD1794, CD1906, CD2046, CD2098, CD2216, CD2264, CD2274, CD2309, CD3188, CD3288, CD3367 and CD2961, or a product of said genes.

### Claim 7 (depends on 1)

The method of claim 1, wherein the coding nucleotide sequence of CD3609 is that of SEQ ID NO:36 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3617 is that of SEQ ID NO: 2 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3618 is that of SEQ ID NO:3 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3635 is that of SEQ ID NO:4 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3638 is that of SEQ ID NO:5 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD0638 is that of SEQ ID NO:21 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD1424 is that of SEQ ID NO:22 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD 1487 is that of SEQ ID NO:23 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD1543a is that of SEQ ID NO:24 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CDI 794 is that of SEQ ID NO:25 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD1906 is that of SEQ ID NO:26 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD2046 is that of SEQ ID NO:27 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD2098 is that of SEQ ID NO:28 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD2216 is that of SEQ ID NO:29 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD2264 is that of SEQ ID NO:30 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD2274 is that of SEQ ID NO: 31 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD2309 is that of SEQ ID NO:32 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3188 is that of SEQ ID NO:33 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3288 is that of SEQ ID NO:34 or a sequence at least 80% identical thereto, the coding nucleotide sequence of CD3367 is that of SEQ ID NO:35 or a sequence at least 80% identical thereto, or the coding nucleotide sequence of CD2961 is that of SEQ ID NO:1 or a sequence at least 80% identical thereto.

### Claim 8 (depends on 1)

The method of claim 1, wherein the presence of one or more of said genes, or products of one or more of said genes, is detected by a primer-directed amplification reaction.

### Claim 9 (depends on 8)

The method of claim 8, wherein said primer-directed amplification reaction is a polymerase chain reaction.

### Claim 10 (depends on 1)

The method of claim 1, wherein the Clostridium difficile is of a ribotype selected from the group consisting of 106, 078, 020, 001, 005, 026, 014, 027, 078v, 015, 015-19, 023, 002, 053 and 140.

### Claim 11 (depends on 1)

The method of claim 1, wherein the presence of said product of said genes is determined by an antibody-mediated detection method.

### Claim 12 (depends on 1)

The method of claim 1 which comprises contacting said sample with a detection moiety which can detect one of said genes or gene products.