Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

Table 1 Standard strains used in the present study

Genus or species	Standard strain ATCC accession no.
S. aureus	26001, 26111, 26113
V. cholerae	16025, 16026, 16028
Shigella spp.	51081, 51207, 51335
E. coli O157:H7	44752, 43889, 43859
V. parahaemolyticus	20502, 20506, 20507
Salmonella spp.	50001, 50004, 50013
Y. enterocolitica	52207, 52211, 52215
L. monocytogenes	54003, 54005, 54006
Brucella spp.	23456
C. botulinum	64201, 64203
B. cereus	63301, 6051, 63509
C. perfringens	64711, 13048

Table 2 Universal primers used in the present study

Primer name	Sequence (5’→3’)	PCR product size (bp)
16SF	CGCTGGCGGCAGGCCTAACACATGC	500
16SR	Biotin-GCGGCTGCTGGCACGGAGTTAGCC
23SF	ACCGATAGTGAACCAGTACCGTGAG	640
23SR	Biotin-TTAAATGATGGCTGCTTCTAAGCC

Table 3 Oligonucleotide probes used in the present study

Probe	Sequence (5’→3’)	Target
1	GGGAGTAAAGTTAATACCTTTGCTCATTGA	Salmonella spp.
2	CACACTGGAACTGAGACACGGTCGAGACTCCTACG GGA	Bacterial universal probe
3	CGTACCATTTGCTACGGAATAACTCAGGGAAACTTGTG	Brucella spp.
4	TCACCCCATAAAAGAGGCTCCCACTGC	E. coli O157:H7
5	TATAAGAGAATCGCATGATTTTCTTATCCAAAGATTTAT	C. botulinum
6	TGCTAGTTGAATAAGCTGGCACCTTGACG	B. cereus
7	ATGGCATCATCATTCAACCATTGGAGCAATCCGCTATGAGATGGACCC	C. perfringens
8	GGTTTCAGGTTCTTTTTCACTCCCCTCGCCG	Common probe for Shigella and Salmonella spp.
9	AAACGAGTTATCTGAACCTTCGGGGAACGATAACGG	V. parahaemolyticus
10	GAAGGCCTTTTCGATAATGATACCGGCGCTCTGCTCTCCC	Shigella spp. and Enteroinvasive E. coli
11	GGTGTTGTGGTTAATAACCGCAGCAATTGA	Shigella spp.
12	CTTCAATAATGCCAGCAGCTCCAACCCCGAAATAGATA	Salmonella typhi
13	CATAAAGGTTAATAACCTTTGTGATTGACGT	Y. enterocolitica
14	GCGGCAGCGGGAAGTAGTTTACTACTTTGCCGG	Yersinia spp.
15	CAGCACAGAGGAACTTGTTCCTTGGGTGGCGAG	V. cholerae
16	TGTTGTTAGAGAAGAACAAGGATAAGAGTAACTGCT	L. monocytogenes
17	ACATATGTGTAAGTAACTGTGCACATCTTGACGGTA	S. aureus
P	TTTGGCTAACTCCGTGCCAGCAGCCGCG	Positive control
C	BIOTIN-CCGCTGTATCACAAGGGCTGGTACCTTT	Color control
N	TTTCCGCTGTATCACAAGGGCTGGTACC	Negtive control
B	0.5 mol/L sodium bicarbonate buffer	Blank control

Table 4 Comparison between the improved RDB and culture

Intestinal pathogen	RDB (+)/culture (+)	RDB (+)/culture (-)	RDB (-)/culture (+)
S. aureus	52	13	12
V. cholerae	4	3	5
Shigella spp.	51	3	3
E. coli O157:H7	28	2	2
V. parahaemolyticus	158	16	11
Salmonella spp.	36	5	3
Y. enterocolitica	3	4	4
L. monocytogenes	4	1	2
B. cereus	3	0	1
C. perfringens	2	0	1
S. aureus and Shigella spp.	2	2	0
S. aureus and Salmonella spp.	2	0	0
Salmonella spp. and Shigella spp.	3	0	0
V. parahaemolyticus and V. cholerae	1	0	0
V. parahaemolyticus and Shigella spp.	5	1	0
Total	354	501	442

¹ was compared with

², χ² = 464, P > 0.05. RDB: Reverse dot blot.