Rapidly and easily acquired information on bacterial and host genes is becoming increasingly important for diagnosis and decision-making when choosing suitable therapies for human infectious diseases. This makes new demands on clinical research and routine laboratories to develop molecularly based methods that can accurately identify and characterise bacterial virulence and host susceptibility and/or resistance genes. The amount of genomic DNA available for such genetic analyses is often limited. Quite often, only minute amounts of bacterial DNA can be found in human biopsies. Moreover, it is desirable to perform concurrent, multiple bacterial and host genotyping analyses from the same, limited amount of biopsy DNA. In such studies, the traditional approach is to purify DNA from cultured bacterial strains isolated from the biopsy specimens . Sample preparation is commonly carried out using time-consuming cartridge or bead-based techniques. These methods do not allow an efficient high-throughput analysis of clinical samples and significant variations of DNA yield and purity can be observed. By contrast, an automated nucleic acid extractor combined with MDA-amplification yields DNA of high purity and integrity that can be used in downstream applications. Indeed, recent studies have demonstrated that PCR amplifications using MDA-amplified DNA can also be carried out under conditions where PCR amplifications normally are hampered due to the presence of PCR inhibitors .
In recent years, MDA has been tried out for the amplification of microbial DNA [36, 48, 49] and total DNA (bacterial and cellular) isolated from human biopsy specimens [38, 39]. This study shows the feasibility of using MDA-amplified total DNA, isolated from human biopsy specimens, for research and clinical diagnostic analysis of both host and infecting bacterium in the same DNA pool.
Pyrosequencing analysis of the16S rDNA variable V3 region revealed the presence of different H. pylori subspecies in the different biopsy specimens which is in agreement with previous reports showing that subtle DNA sequence variations occur in the 16S rDNA variable V3 region of H. pylori, providing a consistent system for subtyping [30, 43]. The taxonomy of these H. pylori strains may be a matter of debate. Subdivision of the species H. pylori into subspecies, based on biotypes, pathotypes or serotypes for taxonomic as well as clinical reasons has been suggested [50, 51].
Pro-inflammatory IL1B and IL1RN polymorphisms are associated with increased risk of gastric carcinoma in Caucasian populations . Carriers of these pro-inflammatory polymorphisms revealed an increased IL1B gene expression pattern [19, 53] in the mucosa and increased prevalence of intestinal metaplasia and atrophic gastritis . Similarly, a genome-wide linkage analysis identified SNPs in IFNGR1 affecting H. pylori infection . Due to the limited number of biopsies analysed, we were not able to draw any statistically significant conclusion regarding allele frequencies in H. pylori-infected and histologically normal individuals. However, the primary goal of the present study was not to perform a clinical study at large but rather to establish new methodological approaches. In analogy with these findings we have recently shown that MDA-DNA derived from minute amounts of archival plasma/serum DNA allowed us to identify cytokine polymorphic SNP-sites by means of pyrosequencing analysis . The use of IL1B-SNP analysis by means of PCR-restriction-fragment-length polymorphism where IL1B-511, IL1B-31 and IL1B+3954 PCR amplicons are digested with restriction enzyme AvaI, TaqI and AluI, respectively, is a widely accepted approach [19–21]. However, restriction enzyme digestion, followed by agarose gelelectrophoresis is a time consuming and, in a clinical routine laboratory context, a tedious process requiring up to 100 ng DNA in each assay . Thus, we conclude that MDA-DNA derived from human biopsy specimens provides a reliable source for cytokine-SNP analysis and, therefore, the same approach may be applied for the characterisation of other host genetic factors in population studies at large
Incorporation of universal M13-sequencetags at the 5'-end of PCR primers facilitated straightforward sequencing of amplicons, which makes culturing of bacteria from human biopsy specimens and cloning of PCR amplicons prior to DNA sequencing unnecessary. This makes it possible for clinical routine laboratories using this technique to rapidly produce sequencing results. Sequencing of M13-tagged amplicons was first described for SPA-typing . The technique has successfully been applied in our laboratory for direct sequencing of PCR amplicons in gene expression studies of vasopressin receptor mRNA splice-variants expressed in the human gastro-intestinal tract and surrounding tissues . In the present study we have used M13 sequence-tags for successful partial sequencing of the H. pylori virulence vacA gene (Fig. 6). Multiple H. pylori strain infection was not seen in this study, although the number of samples included is small. Primer design is of utter importance since species-specific primers are essential to gain pure and reliable sequencing results directly from DNA isolated from a mixed flora.
According to a recent study, the intermediate region (i-region) of the H. pylori vacA gene is an important and independent marker of VacA-associated pathogenicity . All i1-type but no i2-type strains induce vacuolation. Chimeric versions of the i-region (i1-i2) can induce low level of vacuolation in vitro. According to this criteria, our results show two tentatively non-toxic strains (s2/i2/m2- No 21 and s1/i2/m2- No 6) and one with reduced toxicity (s1/i1-i2/m2 No 14; No 18 is discussed below). The remaining biopsies contain toxic H. pylori s1/i1/m1 strains (Table 4).
False negative PCR results from the multiplex-PCR (Table 4) were revealed when using new primers, targeting conserved regions of the vacA gene. The negative results were caused by deletions in the RHM region, yielding a smaller amplicon than expected by previous analysis (Fig. 6). One sample (No 18) had large deletions in the RHM region of the gene, leaving out vacA-primer binding sites commonly used in vacA PCR amplification assays (Fig. 1 and 6). The sequence contained several premature stop codons downstream of the deletion (data not shown), indicating a possible inactive form of VacA. However, further analysis is needed in order to establish if this strain can produce an active protein.
The conclusions to be drawn from the present data is that the choice of primers in vacA PCR amplification assays influences the apparent prevalence vacA-positive H. pylori strains and, hence, precaution has to be taken in the interpretation of vacA-negative PCR amplification results. Thus, our results are in good agreement with previous reports that revealed a high level of vacA genotype variation such as single nucleotide mutations, deletion and in frame stop codons in the vacA alleles of non-toxic H. pylori strains [14, 15], but it seems questionable to what extent published vacA PCR amplification results can be compared between studies if DNA sequencing is not performed. From our and other studies it seems evident that it is necessary to establish partial DNA sequences from either vacA PCR amplicons (Fig. 3, 5, 6) or from full-length vacA open reading frames. Thus, high-throughput sequencing of M13 sequence-tagged PCR amplicons appears to be a more promising approach, both in research and clinical routine laboratories. This principle could be applied to any situation in which sequencing of PCR amplicons is desirable and thereby, one could abstain from tedious amplicon cloning and plasmid preparation procedures prior to DNA sequence analysis.
Histology is considered a sensitive method for detection of H. pylori in gastric biopsies. However, a recent study has demonstrated that PCR amplification using H. pylori-specific primers detected H. pylori-DNA in histological-negative gastric biopsies, indicating the clinical relevance of H. pylori detection by PCR amplification in biopsies with characteristic inflammatory changes . Similarly, Kisa and co-workers  evaluated different diagnostic methods for the detection of H. pylori in gastric biopsy specimens. In their study the authors concluded that nested PCR amplification assays are necessary to detect H. pylori-DNA in gastric biopsy specimens. From this study we conclude that MDA-amplified DNA derived from minute amounts of biopsy specimen DNA is well suited for PCR-amplification and subsequent sequencing of M13 sequence-tagged amplicons. Thus, it is possible to avoid nested PCR amplification assays which often have to be combined with Southern blot analysis to increase the sensitivity and specificity [28, 30].