Development of a Multiplex PCR Technique for Detection and Epidemiological Typing of Salmonella in Human Clinical Samples

Abstract

We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:−. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.

Salmonella enterica is one of the major bacterial agents that cause foodborne infections in humans all over the world (4). In Spain, the most important serotypes causing disease are Salmonella enterica serotypes Enteritidis, Typhimurium, Hadar, and subsp. I serotype 4,5,12:i:− (18). Traditional Salmonella detection methods are based on cultures using selective media and characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming. Therefore, a rapid method is necessary for identification of Salmonella serotypes from clinical specimens. There are several PCR assays to detect Salmonella bacteria in feces, but diagnostic PCR is limited by the presence of inhibiting substances in complex biological samples (7, 11). These substances can interfere with cell lysis or inactivate the DNA polymerase, and DNA extraction procedures are usually necessary to remove them (2). The utility of multiplex PCR as a tool for pathogen detection in clinical and environmental samples is well documented (10, 15, 19).

The aim of this study was to develop a multiplex PCR assay able to detect Salmonella and simultaneously detect the five most important serotypes and phage types in Spain. This technique should be able to avoid the effect of the PCR inhibitors in clinical samples and had to be reliable, simple, and accurate.

We designed new PCR primers using previously described genetic targets (Table 1) and the Jellyfish primer design software (BioWare Corp., Edmonton, Alberta, Canada). The primer set (synthesized by Invitrogen, Paisley, United Kingdom) was chosen to amplify products with lengths similar to those of the 100-bp ladder (Amersham Biosciences, Piscataway, N.J.) bands. A total of six different sequences were amplified in each reaction mixture: a Salmonella genus-specific sequence (204 bp), a serotype Enteritidis-specific sequence (304 bp), a serotype Typhimurium-specific sequence (401 bp), a sequence specific for serotype Typhimurium DT104 and U302 (102 bp), a Salmonella C2 serogroup-specific sequence (502 bp), and a sequence specific for serotype 4,5,12:i:− (705 bp).

TABLE 1.

Primer sequences and sizes of PCR-amplified targets

Primer	Sequence (5′→3′)	Amplification target	Amplicon length (bp)	Assigned no.a	Primer concn (nM)	Reference
104F	ATG CGT TTG GTC TCA CAG CC	Salmonella serotype Typhimurium DT104 and U302	102	1	100	17
104R	GCT GAG GCC ACG GAT ATT TA				75
OMPCF	ATC GCT GAC TTA TGC AAT CG	Salmonella genus	204	2	50	8
OMPCR	CGG GTT GCG TTA TAG GTC TG				50
ENTF	TGT GTT TTA TCT GAT GCA AGA GG	Salmonella serotype Enteritidis	304	4	75	1
ENTR	TGA ACT ACG TTC GTT CTT CTG G				100
TYPHF	TTG TTC ACT TTT TAC CCC TGA A	Salmonella serotype Typhimurium	401	8	100	16
TYPHR	CCC TGA CAG CCG TTA GAT ATT				100
HADF	ACC GAG CCA ACG ATT ATC AA	Salmonella serogroup C2	502	16	100	13
HADR	AAT AGG CCG AAA CAA CAT CG				100
4512F	CGC TGT GGT GTA GCT GTT TC	Salmonella serotype 4,5,12:i:−	705	32	100	3
4512R	TCT GCC ACT TCT TCA CGT TG				100
IC-Fb	atg cgt ttg gtc tca cag ccT TCA TTT CAG CAT TTA TTG GTT GT	IC DNA	990		400	This study
IC-Rb	tga act acg ttc gtt ctt ctg gGC TTT TCT AAT TTA ACC TTT GTC AGG				400

^aNumber assigned to the band to obtain the profile code.

^bPrimers used to create the chimeric IC. The 3′ ends (in capital letters) were designed to amplify a fragment within the lambda phage sequence, and the 5′ ends (in lowercase letters) were the 104F and ENTR sequences.

An artificially created chimeric DNA was used as an internal control (IC) in every reaction mixture. This IC was used to detect inhibition in the PCR due to the presence of inhibitory substances in the clinical samples. The IC was designed according to a strategy previously described (6) slightly modified to obtain a 990-bp fragment and consisted of a fragment (948 bp) of a region of the lambda phage flanked by two of the primers used in the multiplex PCR (104F and ENTR).

Every amplification profile was coded with a number obtained by the addition of the values corresponding to each amplified band (Table 1). The template DNA for multiplex PCR was prepared as previously described (12). All the PCRs were performed in a final volume of 25 μl in a Robocycler 96 Grad (Stratagene, La Jolla, Calif.). The optimized PCR mixture consisted of 1.5 mM MgCl₂, 200 μM each of the four deoxynucleoside triphosphates (Amersham Biosciences), 1 U of Taq polymerase (Amersham Biosciences), and 60 pmol of IC DNA per sample. Primer sequences and concentrations are given in Table 1. The PCR protocol consisted of the following steps: (i) an initial denaturation step of 2 min at 95°C; (ii) 30 cycles, with 1 cycle consisting of 1 min at 95°C, 1 min at 57°C, and 2 min at 72°C; and (iii) a final elongation step of 5 min at 72°C. The PCR products were electrophoresed in 2.5% (wt/vol) D-1 agarose (Pronadisa, Madrid, Spain), stained with 2 μg of ethidium bromide (Sigma-Aldrich, Madrid, Spain) per ml, and photographed under UV light. In each PCR run, a nontemplate control was included to detect possible external DNA contamination.

A total of 138 microbial strains isolated from veterinary, environmental, food, and clinical sources from Spain, Denmark, and England were used for selectivity determination. These strains were well characterized in terms of genus, species, serotype, and phage type (Table 2). Figure 1 shows the amplification profiles and codes obtained in the technique validation stage. The detection limits of this multiplex PCR were 6,500 CFU of Salmonella serotype Typhimurium LT2 strain and 1 ng of DNA. In our validation study, all the tested Salmonella strains showed at least the 200-bp band (code of ≥2) (Table 2), while none of the non-Salmonella strains showed this band (code of 0). The inclusivity and exclusivity were 100% (14), and the overall agreement (5) for Salmonella bacteria was therefore complete.

TABLE 2.

Strains tested and multiplex PCR results obtained in the optimization and validation experiments

Species tested	Subspecies	Serogroup	Serotype	Phage type	No. of isolates tested	PCR results (bp)a						Type codeb
						102 [1]	204 [2]	304 [4]	401 [8]	502 [16]	705 [32]
Citrobacter freundii					1							0
Enterobacter cloacae					1							0
Enterococcus faecalis					1							0
Escherichia coli					4							0
Hafnia alvei					1							0
Klebsiella pneumoniae					1							0
Proteus vulgaris					1							0
Pseudomonas aeruginosa					1							0
Shigella boydii					1							0
Salmonella enterica	I	B	Bredeney		1		+					2
			Agona		1		+					2
			Brandenburg		1		+					2
			Heidelberg		1		+					2
			4,12:b:−		1		+					2
		C1	Braenderup		1		+					2
			Virchow		2		+					2
			Infantis		1		+					2
		D	Miami		2		+					2
		E	Anatum		1		+					2
		F	11:1,v:−		1		+					2
		G	Cubana		1		+					2
Salmonella enterica	II	K	18:z10:z6		1		+					2
		Y	48:k:e,n,x,z15		1		+					2
Salmonella enterica	IIIa	X	47:z4,z23:−		1		+					2
		Y	48:z4,z23:−		1		+					2
		Y	48:−:−		1		+					2
Salmonella enterica	IIIb	X	47:i:z		1		+					2
		Y	48:r:z		1		+					2
		58	58:r:z		1		+					2
			58:z52:z		1		+					2
		Z	50:i:z		1		+					2
Salmonella enterica	IV	H	6,14:z4,z23:−		2		+					2
		I	16:z4,z32:−		1		+					2
		Y	48:z4,z32:−		1		+					2
Salmonella enterica	I	B	Abony		1	+	+					3
		D	Dublin		1	+	+					3
	IIIb	60	60:r:e,n,x,z15		1	+	+					3
Salmonella enterica	I	D	Enteritidis	Several	34		+	+				6
Salmonella enterica	I	B	Typhimurium	Several	16		+		+			10
Salmonella enterica	I	B	Typhimurium	104	15	+	+		+			11
				12	2	+	+		+			11
		C2	Lindenburg		1	+	+		+			11
Salmonella enterica	I	C2	Blockley		1		+			+		18
			Fayed		1		+			+		18
			Glostrup		1		+			+		18
			Goldcoast		1		+			+		18
			Hadar		2		+			+		18
			Litchfield		2		+			+		18
			Manhattan		1		+			+		18
			Muenchen		1		+			+		18
			Newport		1		+			+		18
Salmonella enterica	I	C2	Chailey		1		+		+	+		26
			Cremieu		1		+		+	+		26
			Duesseldorf		1		+		+	+		26
			Kottbus		1		+		+	+		26
			Hadar		1		+		+	+		26
Salmonella enterica	I	B	4,5,12:i:−	U302	10	+	+		+		+	43

^aThe PCR result, the presence (+) of 102-, 204-, 304-, 401-, 502-, and 705-bp bands, which were assigned numbers 1, 2, 4, 8, 16, and 32 (shown in brackets), respectively, is shown for the strains tested.

^bThe type code is obtained by adding all the assigned numbers to the positive amplicons. Each code is specific for a particular band pattern.

FIG. 1.

Multiplex PCR amplification profiles. All the different multiplex PCR amplification profiles found in this study are shown. The type code is shown above the lanes. The M lanes contain the 100-bp molecular size ladder marker.

Analyzing the PCR profiles for epidemiological purposes, all the serotype Enteritidis strains showed the amplification profile code 6. The serotype Typhimurium strains showed the amplification profile code 10 or 11 (if DT104). The serogroup C2 strains showed profile code 18 or 26, depending on the presence of a band of 401 bp, which was not completely reproducible in some strains. Fortunately, all these strains also presented the serogroup C2-specific band. The serotype 4,5,12:i:− strains showed profile code 43. A serotype Lindenberg strain showed profile code 11. Serotype Lindenberg has the same antigenic formula as serotype Typhimurium, except it has C2 serogroup O antigens. Two serotype Typhimurium DT12 strains also presented profile code 11. In a previous pulsed-field gel electrophoresis typing study (9), the pulsed-field gel electrophoresis profile of one of these isolates was identical to the profile of DT104 strains with three restriction enzymes. Also, a DT104 strain showed profile code 10. In the same study (9), the band pattern of this strain was different from the prevalent pattern of the DT104 isolates. Assay reproducibility was measured using independent strains replicated during the validation period of the technique and reached 91.5%. A 400-bp band was not always found for the Salmonella serogroup C2 strains.

Salmonella detection by both conventional culture and multiplex PCR methods was then performed on 120 consecutive human stool samples obtained at the Basurto Hospital, Bilbao, Spain. A swab soaked with human stool was used to inoculate selenite-cystine broth and allowed to grow overnight at 37°C for Salmonella detection by both techniques. In the conventional culture technique, XLD, MacConkey, and Hektoen agars were inoculated with the incubated broth. Suspicious Salmonella colonies were confirmed with triple sugar iron (TSI) agar, API 20E strips (BioMèrieux, Marcy l'Etoile, France), and serotyped according to the Kauffman-White scheme. For Salmonella detection by multiplex PCR, 100 μl of the incubated broth was diluted in 10 ml of fresh broth and incubated for 4 h at 37°C. The broth was then centrifuged at 4°C at 3,000 × g, and the pellet was washed with phosphate-buffered saline. The cells were centrifuged again and resuspended in 200 μl of water. The bacterial DNA was extracted by boiling as described previously (12). A 5-μl aliquot of the supernatant was used as template DNA in the multiplex PCR described above. In addition, a final concentration of 10% (wt/vol) polyethylene glycol (Sigma-Aldrich) was used as a PCR facilitator in the reaction mixture. One hundred seven of the samples gave coincident results by both techniques (Table 3). The multiplex PCR results for two samples were considered false-negative results, and one sample was detected as belonging to the Salmonella genus instead of serotype Enteritidis. In 10 samples (8%), PCR inhibition occurred, including one serotype Enteritidis strain that was isolated by culture. The sensitivity value of the technique was 93%, the specificity was 100%, and the efficiency was 98%. Cohen's kappa index was 0.95, which indicates high agreement between the two techniques.

TABLE 3.

Comparison of results obtained analyzing human stool samples by multiplex PCR and by culture and serotyping

Result by culture and serotyping	No. of samples in the following category by Multiplex-PCR
	Inhibitiona	Negative	Type code 2b	Type code 6	Type code 10	Type code 11	Total
Negative	9	81					90
Salmonella serotype Enteritidis	1	2c	1d	22			26
Salmonella serotype Typhimurium					2	2	4
Total	10	83	1	22	2	2	120

^aInhibition of multiplex PCR.

^bThe type code is obtained by adding all the assigned numbers to the positive amplicons. Each code is specific for a particular band pattern.

^cFalse-negative data in multiplex PCR.

^dMistaken result in multiplex PCR.

In this article, we have described a multiplex PCR-based diagnosis method for Salmonella that is simple, inexpensive, and sensitive and enables the quick and precise detection of the most prevalent serotypes of Salmonella in human clinical samples. Although our results are preliminary, this PCR assay would offer an effective alternative to traditional typing methods for the identification and differentiation of the most clinically relevant Salmonella types.