Method for determining the presence of diarrhoea causing pathogens

1. A method for determining the presence of diarrhoea causing pathogens in a biological sample comprising the steps of:

i) performing a multiplex polymerase chain reaction on the biological sample or nucleic acid isolated therefrom with multiplex primer pairs, wherein the multiplex primer pairs comprise ETEC primer pairs that amplify a nucleic acid sequence (g) of the ETEC amplicon consisting of SEQ ID NO:61, a nucleic acid sequence (h) of the ETEC amplicon consisting of SEQ ID NO:62, and a nucleic acid sequence (i) of the ETEC amplicon consisting of SEQ ID NO:63, wherein each of said multiplex primer pairs recognizes and binds to one of said ETEC amplicons consisting of SEQ ID NO:61, 62 or 63, and wherein the multiplex primer pairs comprise Campylobacter primer pairs that amplify a nucleic acid sequence (k) of the Campylobacter amplicon consisting of SEQ ID NO:65, and a nucleic acid sequence (1) of the Campylobacter amplicon consisting of SEQ ID NO:66; and

ii) detecting for amplification of the nucleic acid sequences (g), (h), (i), (k) and (l), wherein the amplification of any of the nucleic acid sequences (g), (h) and (i) is indicative of the presence of diarrhoea causing pathogen ETEC in the biological sample, and wherein the amplification of any of the nucleic acid sequences (k) and (l) is indicative of the presence of diarrhea causing pathogen Campylobacter in the biological sample.

2. The method according to claim 1 , wherein step i) further comprises primer pairs which amplify each of the Yersinia amplicons comprising SEQ ID NOS: 67-68.

3. The method according to claim 1 , wherein in step i) the sample or isolated nucleic acid therefrom is contacted with at least one ETEC primer pair selected from the group consisting of a nucleic acid sequence (g1) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:13 and a nucleic acid sequence (g2) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:14; a nucleic acid sequence (h1) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:15 and a nucleic acid sequence (h2) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:16; and a nucleic acid sequence (i1) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO: 17 and a nucleic acid sequence (i2) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:18.

4. The method according to claim 1 , wherein said multiplex primer pairs are selected from the group consisting of a nucleic acid sequence (k1) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:21 and a nucleic acid sequence (k2) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:22; and a nucleic acid sequence (l1) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:23 and a nucleic acid sequence (l2) of at least 10 consecutive nucleotides of the sequence consisting of SEQ ID NO:24.

5. The method according to claim 1 , wherein in step i) the sample or isolated nucleic acid therefrom is contacted with further primer pairs comprising at least one of the following sequences or a primer consisting of at least 10 contiguous nucleotides present in at least one of the following sequences:

A) forward primer:

(SEQ ID NO: 1)

5′-GCGTTCTTATGTAATGACTGCTGAAG-3′,

reverse primer:

(SEQ ID NO: 2)

5′-AGAAATTCTTCCTACACGAACAGAGTC-3′;

B) forward primer:

(SEQ ID NO: 3)

5′-TGCATCCAGAGCAGTTCTGC-3′,

reverse primer:

(SEQ ID NO: 4)

5′-CGGCGTCATCGTATACACAGG-3′;

C) forward primer:

(SEQ ID NO: 5)

5′-CCAGGCTTCGTCACAGTTGC-3′,

reverse primer:

(SEQ ID NO: 6)

5′-CAGTGAACTACCGTCAAAGTTATTACC-3′;

D) forward primer:

(SEQ ID NO: 7)

5′-GCTCTTCGGCACAAGTAATATCAAC-3′,

reverse primer:

(SEQ ID NO: 8)

5′-TCTATTTTAAATTCCGTGAAGCAAAACG-3′;

E) forward primer:

(SEQ ID NO: 9)

5′-TGGTCCATCAGGCATCAGAAGG-3′,

reverse primer:

(SEQ ID NO: 10)

5′-GGCAGTGCGGAGGTCATTTG-3′;

F) forward primer:

(SEQ ID NO: 11)

5′-TGTCTTTATAGGACATCCCTGATACTTTC-3′,

reverse primer:

(SEQ ID NO: 12)

5′-TATCTACTCTTGATGCCAGAAAACTAGC-3′;

J) forward primer:

(SEQ ID NO: 19)

5′-GGAAGCAATACATATCTTAGAAATGAACTC-3′,

reverse primer:

(SEQ ID NO: 20)

5′-TCGGACAACTGCAAGCATCTAC-3′;

M) forward primer:

(SEQ ID NO: 25)

5′-GTTTGGTACAGTTTATGGCATTTCAC-3′,

reverse primer:

(SEQ ID NO: 26)

5′-CATGGCAATATCAACAATACTCATCTTAC-3′;

N) forward primer:

(SEQ ID NO: 27)

5′-CAGGAGCATGAGGTTCACAGTATG-3′,

reverse primer:

(SEQ ID NO: 28)

5′-TCTCTGGCCCCGCACAATG-3′;

O) forward primer:

(SEQ ID NO: 29)

5′-GGGCTACAGAGATAGATATTACAGTAACTTAG-3′,

reverse primer:

(SEQ ID NO: 30)

5′-CCACGGCTCTTCCCTCCAAG-3′;

P) forward primer:

(SEQ ID NO: 31)

5′-TTCCGGTCGATCCTGCC-3′,

reverse primer:

(SEQ ID NO: 32)

5′-GTTGTCCTGAGCCGTCC-3′;

Q) forward primer:

(SEQ ID NO: 33)

5′-AGACGATCCAGTTTGTATTAG-3′,

reverse primer:

(SEQ ID NO: 34)

5′-GGCATCCTAACTCACTTAG-3′;

and

R) forward primer:

(SEQ ID NO: 35)

5′-TCTGGAAAACAATGTGTTC-3′,

reverse primer:

(SEQ ID NO: 36)

5′-GGCATGTCGATTCTAATTC-3′.

6. The method according to claim 3 , wherein the presence of the amplified target C sequence, of each of primer pairs in the PCR reaction indicates the presence of diarrhoea causing pathogens in the sample in the following way:

the product of primer pair A) or B) indicates the presence of EHEC;

the product of primer pair C) indicates the presence of EHEC/EPEC;

the product of primer pair D) indicates the presence of Salmonella;

the product of primer pair E) or F) indicates the presence of Shigella /EIEC;

the product of primer pair G), H), or I) indicates the presence of ETEC;

the product of primer pair J) indicates the presence of EAEC;

the product of primer pair K) indicates the presence of Campylobacter jejuni;

the product of primer pair L) indicates the presence of Campylobacter coli;

the product of primer pair M) indicates the presence of Yersinia enterocolitica/pseudotuberculosis;

the product of primer pair N) indicates the presence of Yersinia pseudotuberculosis/pestis;

the product of primer pair O) indicates the presence of Vibrio cholerae:

the product of primer pair P) indicates the presence of Giardia lamblia;

the product of primer pair Q) indicates the presence of Entamoeba histolytica ; and

the product of primer pair R) indicates the presence of Cryptosporidium sp.

7. The method according to claim 1 , wherein said biological sample is a stool sample or a food sample.

8. The method according to claim 5 , wherein said multiplex PCR assay is performed as a real-time polymerase chain reaction and probes consisting of at least 10 contiguous nucleotides present in the following sequences are specifically used with each of primer pairs:

the probe for primer pair A):

(SEQ ID NO: 37)

5′-TCCATGATARTCAGGCAGGACACTACTCAACCTTCC-3′,

the probe for primer pair B):

(SEQ ID NO: 38)

5′-TTGTCACTGTCACAGCAGAAGCCTTACGC-3′,

the probe for primer pair C):

(SEQ ID NO: 39)

5′-AGATTAACCTCTGCCGTTCCATAATGTTGTAACCA-3′,

the probe for primer pair D):

(SEQ ID NO: 40)

5′-CCAAACCTAAAACCAGTAAAGGCGAGCAGC-3′,

the probe for primer pair E):

(SEQ ID NO: 41)

5′-TCACTCCCGACACGCCATAGAAACGCATTT-3′,

the probe for primer pair F):

(SEQ ID NO: 42)

5′-ACAAACAGCAAAAGAGCATAGCATCCGAGAACT-3′,

the probe for primer pair G):

(SEQ ID NO: 43)

5′-CAAATATCCGTGAAACAACATGAC-3′,

the probe for primer pair H):

(SEQ ID NO: 44)

5′-AGGATTACAACACAATTCACAGCAGT-3′,

the probe for primer pair I):

(SEQ ID NO: 45)

5′-AGCAGGTTTCCCACCGGATCACCA-3′,

the probe for primer pair J):

(SEQ ID NO: 46)

5′-TCCGTATATTATCATCAGGGCATCCTTTAGGCGT-3′,

the probe for primer pair K):

(SEQ ID NO: 47)

5′-AAGACCCACAGTTTTACCAAGTTTT-3′,

the probe for primer pair L):

(SEQ ID NO: 48)

5′-AACTTGGCTCTTCTTATGTGCGT-3′,

the probe for primer pair M):

(SEQ ID NO: 49)

5′-CCTGGATAAGCGAGCGACGTATTCTCTATGC-3′,

the probe for primer pair N):

(SEQ ID NO: 50)

5′-AAACCAAAGCCGCCCACACCACAG-3′,

the probe for primer pair O):

(SEQ ID NO: 51)

5′-AACCTGCCAATCCATAACCATCTGCTGCTG-3′,

the probe for primer pair P):

(SEQ ID NO: 52)

5′-ACGAAGCCATGCATGCCCGCT-3′,

the probe for primer pair Q):

(SEQ ID NO: 53)

5′-ACAAAATGGCCAATTCATTCAATGAA-3′,

the probe for primer pair R):

(SEQ ID NO: 54)

5′-CCTCCTAATCCAGAATGTCCTCCAG-3′,

wherein the probes comprise modified nucleotides increasing melting temperature, Tm, of the probes.

9. The method according to claim 5 , wherein primer pairs A) to F) and G) to N) are in separate PCR reactions.

10. The method according to claim 5 , wherein the method comprises the following PCR reactions: the first reaction with primer pairs A) to D), the second reaction with primer pairs K) to L), the third reaction with primer pairs E), F), M), N) and O) and the fourth reaction with primer pairs P) to R).

11. The method according to claim 5 , wherein the method comprises the following PCR reactions: the first reaction with primer pairs A) to D), the second reaction with primer pairs G) to I) and K) to N), the third reaction with primer pairs E), F), J) and O) and the fourth reaction with primer pairs P) to R).

12. The method according to claim 3 , wherein the at least one ETEC primer pair comprises a primer consisting of SEQ ID NO: 13, 14, 15, 16, 17 or 18.

13. The method according to claim 12 , wherein the at least one ETEC primer pair consists of the primers consisting of SEQ ID NOS: 13 and 14.

14. The method according to claim 12 , wherein the at least one ETEC primer pair consists of the primers consisting of SEQ ID NOS: 15 and 16.

15. The method according to claim 12 , wherein the at least one ETEC primer pair consists of the primers consisting of SEQ ID NOS: 17 and 18.

16. The method according to claim 4 , wherein the at least one Campylobacter primer pair comprises a primer consisting of SEQ ID NO: 21, 22, 23 or 24.

17. The method according to claim 16 , wherein the at least one Campylobacter primer pair consists of the primers consisting of SEQ ID NOS: 21 and 22.

18. The method according to claim 16 , wherein the at least one Campylobacter primer pair consists of the primers consisting of SEQ ID NOS: 23 and 24.

19. The method according to claim 5 , wherein said multiplex PCR assay is performed as a real-time polymerase chain reaction and probes comprising the following sequences are specifically used with each of primer pairs:

the probe for primer pair A):

(SEQ ID NO: 37)

5′-TCCATGATARTCAGGCAGGACACTACTCAACCTTCC-3′,

the probe for primer pair B):

(SEQ ID NO: 38)

5′-TTGTCACTGTCACAGCAGAAGCCTTACGC-3′,

the probe for primer pair C):

(SEQ ID NO: 39)

5′-AGATTAACCTCTGCCGTTCCATAATGTTGTAACCA-3′,

the probe for primer pair D):

(SEQ ID NO: 40)

5′-CCAAACCTAAAACCAGTAAAGGCGAGCAGC-3′,

the probe for primer pair E):

(SEQ ID NO: 41)

5′-TCACTCCCGACACGCCATAGAAACGCATTT-3′,

the probe for primer pair F):

(SEQ ID NO: 42)

5′-ACAAACAGCAAAAGAGCATAGCATCCGAGAACT-3′,

the probe for primer pair G):

(SEQ ID NO: 43)

5′-CAAATATCCGTGAAACAACATGAC-3′,

the probe for primer pair H):

(SEQ ID NO: 44)

5′-AGGATTACAACACAATTCACAGCAGT-3′,

the probe for primer pair I):

(SEQ ID NO: 45)

5′-AGCAGGTTTCCCACCGGATCACCA-3′,

the probe for primer pair J):

(SEQ ID NO: 46)

5′-TCCGTATATTATCATCAGGGCATCCTTTAGGCGT-3′,

the probe for primer pair K):

(SEQ ID NO: 47)

5′-AAGACCCACCGTTTTACCAAGTTTT-3′,

the probe for primer pair L):

(SEQ ID NO: 48)

5′-AACTTGGCTCTTCTTATGTGCGT-3′,

the probe for primer pair M):

(SEQ ID NO: 49)

5′-CCTGGATAAGCGAGCGACGTATTCTCTATGC-3′,

the probe for primer pair N):

(SEQ ID NO: 50)

5′-AAACCAAAGCCGCCCACACCACAG-3′,

the probe for primer pair O):

(SEQ ID NO: 51)

5′-AACCTGCCAATCCATAACCATCTGCTGCTG-3′,

the probe for primer pair P):

(SEQ ID NO: 52)

5′-ACGAAGCCATGCATGCCCGCT-3′,

the probe for primer pair Q):

(SEQ ID NO: 53)

5′-ACAAAATGGCCAATTCATTCAATGAA-3′,

the probe for primer pair R):

(SEQ ID NO: 54)

5′-CCTCCTAATCCAGAATGTCCTCCAG-3′,

wherein the probes comprise modified nucleotides increasing melting temperature, Tm, of the probes.