Acknowledgments We thank the reviewers for their constructive

We thank the reviewers for their constructive comments on improving the quality of this paper. This work was supported in part by the National Natural Science Foundation of China (11401519).

A large number of studies confirmed that Helicobacter spp. have their share in the pathogenesis of diseases of the digestive system, ever since Helicobacter pylori was first recognized as an important factor in the genesis of gastritis, peptic ulcer and gastric adenocarcinoma in human (Parsonnet et al., 1991; Dunn et al., 1997). The number of Helicobacter spp. identified is increasing as more research is done in this field. With 34 Helicobacter species now formally named according to LPSN (, it is clear that Helicobacter species can infect human and various animal hosts, as well as colonize different anatomical regions of the gastrointestinal system (Fox, 2002; Recordati et al., 2009).
Culture of helicobacter organisms can be used to identify infected hosts. However, the bacteria are fastidious and initial in vitro isolation therefore requires special microaerophilic conditions and 5–7days (Fox et al., 1994, 1995). Detection of helicobacters by culture is further complicated by the presence of normal flora, since the preferred site for isolation of the bacteria is the gastrointestinal tract. In addition to culture, the immunological assays are also important techniques. However, the difficulties to culture many fastidious Helicobacter species to obtain enough Mifepristone for the extraction of cell surface proteins are still a challenge. Therefore there is a need to develop an easy to use and reliable assay to detect and discriminate helicobacters (Wadstrom et al., 2009).
The association between the Helicobacter spp. and gastrointestinal diseases can also be determined by proving the presence of the Helicobacter spp. DNA in samples that are collected during the examinations. The procedure became simpler and more precise, which allowed an evolution of PCR in the sense of being more feasible, but also a sophisticated and reliable method that has proven its usefulness in dealing with the association. (Engstrand et al., 1992; Lin et al., 1995; Fox et al., 1998; Myung et al., 2000; Monstein et al., 2002). However, Poynter et al. reported that three out of six different commercial laboratories performing helicobacter testing on the same spiked fecal samples failed to detect and identify Helicobacter hepaticus due to the differences in the design of their PCR assays (Poynter et al., 2009). Bulajic et al. concluded that further investigation of Helicobacter spp. role in the development of malignancies and other diseases requires targeting of new genome sequences or design of more specific primers (Bulajic et al., 2012). The PCR subtypes used included nested PCR and single-round PCR. Nested PCR has been used frequently in recent years due to its higher specificity. The most sensitive and widely used method for detecting helicobacter infections is PCR targeting at a genus-specific and conserved region of 16S rRNA (Battles et al., 1995; Bulajic et al., 2012). In this study, we have tried to establish and optimize a hemi-nested PCR assay that would simultaneously detect and differentiate 34 of the formally named helicobacters from feces samples. The primers were designed based on the sequences of 16S rRNA.

Materials and methods

The nested PCR primers were designed to the target 16S rRNA gene specific to all 34 type strains of Helicobacter spp., not including any rare bases. Theoretically, the primers, being able to detect all the type strains, have not any faults according to the Oligo7.0 Software evaluation. In comparison with it, not only the inclusivity but also some faults lies in other primers usually used in the literature, as described in Table 1. Furthermore, the expected products size and optimal annealing temperature to 34 type strains were described in Table 2. Numbers in the sixth column indicate the positions of the primers to different type strains. The optimal annealing temperature fluctuates from 55.3°C to 57.8°C for the inner primers, and from 56.5°C to 57.9°C for the outer primers (Table 2). In practice, the excellent validity of the method was examined by feces simulated samples (Fig. 2), BLAB/c mice infection models (Fig. 3) and H. pylori infection fecal samples (Fig. 4).