Evaluation of 16S rRNA Gene-Based PCR Assays for Genus-Level Identification of Helicobacter Species

Abstract

The inclusivity, exclusivity, and detection limit of six 16S rRNA gene-based Helicobacter genus-specific PCR assays were examined. Five out of six assays were 100% inclusive, but the tests varied considerably in their exclusivity (9.1 to 95.5%). The clinical detection limit varied between 10³ and 1 viable bacterial cell per reaction mixture.

Currently, at least 9 gastric and 21 enterohepatic formally named Helicobacter species have been identified in a large variety of animal species and in humans (http://www.bacterio.cict.fr/h/helicobacter.html). Infections with these bacteria may interfere with results obtained from experimental research in animals and, thus, may lead to the misinterpretation of the data (8, 12). Therefore, infected animals must be identified. Until now, several Helicobacter genus-specific PCR assays for use in large-scale screening programs have been described (1, 3, 4, 5, 8, 9). The inclusivity and exclusivity of some of these assays has been examined before (3, 5, 8), but the basis of these evaluations differed considerably, particularly with respect to the numbers and choices of strains used to evaluate the tests. This makes an objective comparison of their efficacy very difficult. In this study, purified DNA of a collection of 43 type and reference strains belonging to different Helicobacter (n = 21), Campylobacter (n = 15), Arcobacter (n = 6), and Wolinella (n = 1) species was used to evaluate the inclusivity, exclusivity, and detection limit of six previously described Helicobacter genus-specific PCR assays (1, 3, 4, 5, 8, 9), all targeting the 16S rRNA gene.

All PCR assays were performed in 25-μl volumes containing 2.5 μl 10× PCR buffer (Invitrogen Life Technologies, Merelbeke, Belgium), 0.25 μl of each primer (Operon, Cologne, Germany), 5 μl of deoxynucleoside triphosphate mix (final concentration, 200 μM; Invitrogen Life Technologies), and 1 μl of DNA template (concentrations ranged between 3 and 200 ng DNA/μl, depending on the species). Volumes of Taq polymerase Platinum (Invitrogen Life Technologies), MgCl₂ (Invitrogen Life Technologies), and DNA-free purified water were used as appropriate for each assay (Table 1). Reaction mixtures were heated for 5 min at 94°C as an initial denatur-ation step. PCR cycling conditions were as described in the original studies (1, 3, 4, 5, 9), with amendments from the study of Riley et al. (8), in which 35 cycles of 30 s of denaturation at 94°C, 60 s of annealing at 53°C, and 90 s of elongation at 72°C were used. All assays were terminated with a 5-min extension period of 72°C and were performed with Mastercycler ep thermocyclers (Eppendorf, Hamburg, Germany). Amplicons were detected by the ethidium bromide staining of electrophoresed samples as described previously (2). All PCR assays were performed in triplicate on three separate occasions. If a positive result was obtained with a species not belonging to the genus Helicobacter in all six assays, the obtained amplicons were purified with a QIAquick PCR purification kit (Qiagen, Venlo, The Netherlands) and sequenced as described before (6) using the appropriate primers (Table 1) to exclude the contamination of the DNA with Helicobacter DNA.

TABLE 1.

Helicobacter genus-specific PCR primers and assay specifications

Target gene (amplicon size, in bp)	Primerb	PCR parameters used				Reference
		MgCl2 concn (mM)	Amt (U/μl) of Taq	Primer annealing temp (°C)	No. of cycles
16S rRNA (375)	F, 5′-CTATGACGGGTATCCGGC-3′	1.5a	0.06a	53a	35a	8
	R, 5′-ATTCCACCTACCTCTCCCA-3′
16S rRNA (399)	F, 5′-AACGATGAAGCTTCTAGCTTGCTAG-3′	1.5a	0.05a	65a	35a	5
	R, 5′-GTGCTTATTCSTNAGATACCGTCAT-3′
16S rRNA (1,200)	F, 5′-GCTATGACGGGTATCC-3′	2.25a	0.02a	55a	35a	9
	R, 5′-ACTTCACCCCAGTCGCTG-3′
16S rRNA (780)	F, 5′-CTATGACGGGTATCCGGC-3′	2.5a	0.05a	55a	30a	1
	R, 5′-CTCACGACACGAGCTGAC-3′
16S rRNA (389)	F, 5′-GTAAAGGCTCACCAAGGCTAT-3′	1.5c	0.05c	63a	35c	4
	R, 5′-CCACCTACCTCTCCCACACTC-3′
16S rRNA (764)	F, 5′-GGCTATGACGGGTATCCGGC-3′	1.5a	0.05a	65a	45a	3
	R, 5′-GCCGTGCAGCACCTGTTTTC-3′

^aThe parameter is as described in the original publication.

^bF, forward; R, reverse.

^cThe parameter was not stated by the original authors; the value shown is an in-house parameter.

A detailed overview of the inclusivity (the percentage of Helicobacter strains correctly identified), exclusivity (100 minus the percentage of strains of the nontarget species giving an amplicon of the correct size), and detection limits of all assays is given in Table 2.

TABLE 2.

Inclusivity, exclusivity, and detection limit of each Helicobacter-genus PCR assay^a

Species and detection parameters	Result for PCR assay described in reference:
	8	4	1	5	3	9
Helicobacter equorum	+	+	+	+	+	+
Helicobacter canadensis	+	+	+	+	+	+
Helicobacter pullorum	+	+	+	+	+	+
Helicobacter pametensis	+	+	+	+	+	+
Helicobacter canis	+	+	+	+	+	+
Helicobacter felis	+	+	+	+	+	+
Helicobacter salomonis	+	+	+	+	+	+
Helicobacter bizzozeronii	+	+	+	+	+	+
Helicobacter hepaticus	+	+	+	+	+	+
Helicobacter bilis	+	+	+	M	+	+
Helicobacter cinaedi	+	+	+	+	+	+
Helicobacter pylori	+	+	+	+	+	+
Helicobacter mustelae	+	+	+	+	+	+
Helicobacter cynogastricus	+	+	+	+	+	+
Helicobacter acinonychis	+	+	+	+	+	+
Helicobacter mesocricetorum	+	+	+	+	+	+
Helicobacter marmotae	+	+	+	+	+	+
Helicobacter cholecystus	+	+	+	+	+	+
Helicobacter trogontum	+	+	+	+	+	+
Helicobacter muridarum	+	+	+	+	+	+
Helicobacter fennelliae	+	+	+	M	+	+
Campylobacter coli	−	+	−	+ (W)	−	−
Campylobacter jejuni	−	+	−	+ (W)	−	+ (W), U
Campylobacter concisus	+	+	−	−	+	+ (W), M
Campylobacter fetus	−	−	−	−	−	U
Campylobacter hyointestinalis	−	+	−	−	+, M	−
Campylobacter lari	+	+	−	−	+	+ (W)
Campylobacter mucosalis	+ (W)	+	−	−	+	+ (W)
Campylobacter sputorum	+	+, U	−	U	+	+
Campylobacter curvus	+	+	M	−	+	+, M
Campylobacter gracilis	−	+ (W)	−	−	+, M	+ (W)
Campylobacter insulaenigrae	−	+	−	−	U	−
Campylobacter hominis	+	+	−	−	+	+
Campylobacter lanienae	−	+	U	−	−	−
Campylobacter helveticus	−	+	−	−	M	−
Campylobacter upsaliensis	−	+	−	−	−	−
Arcobacter butzleri	+	+	−	+ (W)	+	+ (W)
Arcobacter cryaerophilus	+	+	−	+ (W)	+	+
Arcobacter skirrowii	+	+	−	−	+	−
Arcobacter cibarius	M	U	−	−	+, M	−
Arcobacter halophilus	−	+	−	−	−	−
Arcobacter nitrofigilis	−	+	−	−	−	−
Wolinella succinogenes	+	+	+	+ (W)	+	+
Inclusivity (%)	100	100	100	90.5	100	100
Exclusivity (%)	54.5	9.1	95.5	77.3	41	50
Analytical detection limitb	2 × 102 (40)	2 × 102 (40)	2 × 102 (40)	2 × 102 (40)	2 × 10 (4)	2 × 10 (4)
Clinical detection limitc	10	1	10	103	1	1

^a+, An amplicon of the expected size was obtained; −, an amplicon was not obtained; + (W), a weak amplicon of the expected size was obtained; U, an amplicon of unexpected size (often weak) was obtained; M, multiple amplicons of unexpected sizes (often weak) were obtained.

^bValues are expressed as femtograms of DNA/reaction mixture. Values in parentheses are the numbers of viable bacterial cells/reaction mixture.

^cValues are expressed as viable bacterial cells/reaction mixture.

Overall, the inclusivity was high for all assays. This may be explained in part by the use of purified DNA extracts as the template. In a study by On and Jordan (7), the use of heated cell lysates was compared to the use of purified DNA, and it was found that the use of cell lysate templates negatively affected the sensitivity of the assays.

All assays performed suboptimally on exclusivity, which ranged between 9.1 and 95.5%. Where applicable, our reexamination of the exclusivity mostly concurred with results obtained by the original authors (3, 5, 9), but usually only very few Campylobacter, Arcobacter, and/or Wolinella strains were included in the original surveys. In general, the investigators chose to include DNA extracts from other bacteria commonly found in the gastric and/or intestinal flora to evaluate the specificity of their assays. Frequently tested organisms were Escherichia coli, Proteus spp., Bacteroides spp., Staphylococcus spp., Streptococcus spp., and Enterococcus spp. Our results emphasize that more problems are encountered with the accurate discrimination of closely related taxa. Therefore, it is important to use a strain collection that adequately reflects the taxonomy of the target species to validate a novel PCR assay.

In all six assays, an amplicon of the correct size was obtained with Wolinella succinogenes DNA. The sequencing of these PCR products yielded fragments that all showed 99 to 100% similarity to the 16S rRNA gene of W. succinogenes ATCC 29543^T. Therefore, the accidental contamination of the Wolinella DNA with Helicobacter DNA leading to false-positive results could be excluded. Phylogenetically, W. succinogenes is very closely related to the genus Helicobacter (11). In view of this, the observed cross-reaction between primers designed to be specific for Helicobacter and Wolinella DNA is not so surprising.

To determine the analytical detection limit of each PCR assay, 10-fold serial dilutions of the genomic DNA of H. pylori ATCC 26695^T (starting from 200 ng DNA/μl) were used as a template in the respective PCR assays, and amplicons were visualized as described above. Additionally, the clinical detection limit of each assay was determined by spiking fecal horse samples, previously tested negative for Helicobacter-related DNA, with decreasing quantities of Helicobacter equorum as previously described (10).

The analytical detection limit was sufficient for all assays and varied between 200 and 20 fg DNA per reaction mixture, which corresponds to approximately 40 and 4 bacterial cells per reaction mixture, respectively (5). For all but one (5) assay, the clinical detection limit coincided with these results (Table 2), thereby demonstrating a good elimination of fecal PCR inhibitors and a good recovery of the target DNA.

It is known that PCR assays that function very specifically by using defined DNA samples as the template may not work properly when more complex samples are studied (3). Therefore, the most accurate assay (1) was tested on an additional 50 DNA extracts from feces of horses infected with H. equorum (6). Helicobacter DNA from each sample was detected with this test. Nonspecific PCR products were obtained from 19 samples (39%), but these fragments of unexpected sizes usually were very weak and therefore did not interfere with the interpretation of the gels.

In the present study, it was demonstrated that the PCR assay described by Al-Soud et al. (1) is especially highly reliable for the genus-level identification of Helicobacter species. This assay is 100% inclusive and 95.5% exclusive, and it can detect as few as 10 bacterial cells per reaction mixture. Moreover, it appeared to be applicable on DNA extracts from fecal samples. Therefore, we recommend using this test for screening fecal samples for the presence of Helicobacter spp.