A global assessment of the source specificity, sensitivity and geographical stability of Bacteroidetes qPCR assays for microbial source tracking

G REISCHER; J HAIDER; N POKORNY; A DE RODA HUSMAN; S WUERTZ; B PRADHAN; C FERGUSON; D BYAMUKAMA; D MUSHI; G GOSHUH; G KO; J EBDON; J ÅSTRÖM; T COAKLEY; R POMA; W AHMED; V VRAJMASU; E TOTH; M SCHADE; A BLANCH; A FARNLEITNER

Isla de Naxos, Grecia

Simposio; 15th International Symposium on Health-Related Water Microbiology; 2009

The need to identify sources of faecal pollution in aquatic environments has led to the development of numerous methods for microbial source tracking (MST). During recent years methods for the detection of source-specific genetic markers mostly targeting the 16S rRNA genes in abundant faecal anaerobes such as Bacteroidetes have become increasingly important to this field. Numerous quantitative PCR (qPCR) assays for the detection of such MST markers have been developed (1-6). However, until now such assays have not been comprehensively tested and compared for their source-specificity and sensitivity beyond the regional level. Therefore, the aim of the presented study was to apply a number of potentially promising assays specific for ruminant or human sources to faecal DNA samples of known origin collected from sites across six continents. The goal was to gain an initial insight into the global applicability of the proposed assays in order to provide recommendations for potential users and to identify goals for future research. In total 320 individual faecal samples of known origin were collected from 15 countries (Argentina, Australia, Austria, Germany, Hungary, Korea, Nepal, Netherlands, Romania, Spain, Sweden, Tanzania, Uganda, United Kingdom, and USA). Of these samples 25% were from humans, 25% from ruminants and 50% from ‘other’ animal sources (e.g. wild birds, pigs, horses, companion animals). Faecal DNA was extracted using commercially available kits. After transfer of the DNAs to Austria, extracts were purified, the DNA concentration and the quality was determined using spectrophotometry. The DNA extracts were subsequently tested for amplifiable faecal DNA and absence of PCR inhibition by applying a general Bacteroidetes specific qPCR assay (3). Only DNAs passing this trial stage were included for further analyses (n =304). The MST assays applied in this study were selected according to the following criteria: i) they targeted human or ruminant/bovine sources; ii) they exhibited good specificity in the original publications; and iii) they were restricted to probe-based 5’-nuclease assays to ensure high marker sensitivity and specificity. Additionally, we included the human-specific SYBRGreen qPCR method published by Seurinck et al. (1), which detects the frequently used Bacteroidetes marker HF183 (7). Six assays were initially included in the investigation (published in 1, 3-6). Implementation of the assays included adaption to the used qPCR equipment and chemistry, preparation of qPCR standard dilution series using plasmids carrying the detected marker sequences and careful evaluation of the assay performance (adjustment of oligonucleotide and Mg2+ concentrations, annealing temperature gradient). The assays passing these trials were then evaluated for source-specificity and sensitivity using the preselected faecal DNA extracts contributed by the collaborating laboratories. The number of false-positives with the human-specific SYBRGreen assay was extremely high, mostly because qPCR measurements were confounded by formation of primer dimers and other unspecific PCR products. This is a well-known weakness of qPCR detection based on SYBRGreen. Melting curve analysis allows determination of nonspecific products to some extent, but all reactions containing such products have to be excluded from analysis. These results corroborated our decision to restrict the choice of methods to probe-based qPCR methods. The source-sensitivity of the tested probe-based assays proved to be fairly high (>80%). On the other hand the source-specificity on the level of faecal samples was often below 80% with strong differences in results between the various countries. However, the average marker abundance in false-positives was usually two to three orders of magnitude lower than in true positive samples. From a methodological point of view it also proved essential to include analysis by gel electrophoresis of products from probe-based qPCR assays when testing source-specificity and sensitivity. In conclusion it was evident that the tested methods for detection of source-specific Bacteroidetes markers do not seem to be applicable on a global scale without additional evaluation. In any case, validation of the used assays at a catchment-scale using faecal samples is a prerequisite for their successful application to new study areas. In each application the level of required specificity is dependent on the prevalence of the potential sources and on the abundance of the respective markers in faeces from those sources in a specific area. The relatively high frequency of false positives with low marker abundance might be an indication that the candidate source-specific Bacteroidetes are sporadically present as transient populations in other hosts, albeit at lower numbers. On the other hand these results might also be attributed to a certain low-level lack of specificity of the applied qPCR methodologies. Additional research is required in order to focus further attention on the issue of host-specificity and qPCR assay performance.