In the present study we developed a pentaplexed real-time PCR assay for the simultaneous detection and quantification of bont/a, bont/b, bont/e, and bont/f, including an internal control IAC in comparison to singleplex real-time PCR assays for all relevant bont target genes. We analyzed the assay performance, specificity, and sensitivity and validated the PCR approaches with differently spiked food samples and with specimens from several cases of botulism.
While the detection of single BoNT genes by conventional or real-time PCR methods is well established, a number of PCR assays for the detection of three or more BoNT genes have also been described (11
). More recent work has complemented these studies by the description and validation of a conventional multiplex approach for the simultaneous detection of all four human pathogenic bont
). Previous approaches have used degenerated primers to detect more than one BoNT gene. Similarly, our pentaplexed PCR assay uses degenerate primers to accommodate sequence variations within the specific binding regions of bont
genes and therefore is able to detect all human pathogenic serotypes and subtypes described thus far (A1, A2, A3, A4, and A5; B1, B2, B3, nonproteolytic B, and bivalent B; E1, E2, E3, E4, E5, and E6; F baratii, proteolytic F, nonproteolytic F, and bivalent F) (5
). In addition, the pentaplexed PCR approach correctly identifies a novel A subtype identified in our group in the context of a recent food-borne botulism case (strain Chemnitz, Table and also unpublished results).
Generally, major advantages of the real-time TaqMan approach compared to the conventional PCR approach (using gel separation of amplified targets) include the increased assay specificity (by a third gene-specific oligoprobe), reduced assay time (online detection of amplification products, no electrophoresis step), and ease of quantification (using defined concentrations of standard plasmids). From a technical perspective, multiplexing a real-time PCR for more than four target genes is challenging for a number of reasons. (i) To date, only a few instruments are able to detect and differentiate more than four different fluorescent reporter dyes, among them the LightCycler 480 II used in the present study. (ii) An increase in the number of primers and probes per PCR at the same time enhances the problem of nonspecific oligonucleotide interactions. Therefore, shorter oligonucleotides with good hybridization performance are necessary. For this purpose, alternative probe chemistries have been introduced, such as MGB- and LNA-containing oligoprobes (19
). (iii) Some of these improved oligoprobes can only be purchased coupled to a limited number of fluorescent dyes (e.g., MGB-containing probes can be purchased only with up to three different reporter dyes).
To our knowledge, the present study is one of the first to describe a multiplex real-time PCR approach that combines both MGB- and LNA-containing oligoprobes within one reaction. Our study describes only the second reported use of a pentaplexed real-time PCR assay, following a recent description of a pentaplexed assay for the simultaneous quantification of respiratory RNA viruses (25
). While setting up our pentaplexed assay, we empirically learned that mixing of MGB- and LNA-containing oligoprobes rendered a superior PCR performance, compared to a multiplex assay with LNA probes only. For each target gene, we carefully selected the optimal probe chemistry (MGB or LNA) and reporter dye (Cy5, VIC, FAM, TEX, and Cyan500) in order to obtain concordant results for the pentaplex approach and the optimal singleplex reaction. In the final pentaplexed assay, both the specificity and the sensitivity were equivalent to the singleplex approach: analysis of 42 C. botulinum
strains and 57 non-C. botulinum
strains using the designed multiplex and singleplex real-time PCR assays showed high specificity for their predicted target, with no observed cross-reactivity.
With respect to the analysis of clinical, environmental, or food samples, the use of an internal amplification control is mandatory for any diagnostic PCR method. An internally controlled PCR would demonstrate failure of the assay, e.g., due to the presence of PCR inhibitors in the complex sample material, and therefore avoids false-negative results (18
). Consequently, the use of an IAC is included in different international standards, e.g., in ISO 22174, for the analysis of food. Among all PCR assays published on the detection of C. botulinum
, however, only a limited number include the use of an IAC (1
). In our pentaplexed PCR approach, the IAC based on a synthetic sequence was simultaneously amplified with the target sequences bont/a
, and bont/f
. In order to avoid competition between the bont
-specific and the IAC-specific amplification, the IAC plasmid pKoMa2 was optimally used at a low concentration (75 copies per reaction). In our description of the multiplex approach, we followed almost all of the recently defined MIQE guidelines for real-time PCR experiments which serve to standardize experimental practice and to promote consistency between laboratories (4
When we tested the sensitivity of the multiplex PCR assay with C. botulinum A, B, E, and F spiked into different food, we found a limit of detection of 103 to 105 CFU/ml directly from the spiked food samples, without prior enrichment by anaerobic culture. In addition, we successfully applied the multiplex and singleplex real-time PCR assays to specimens from cases of food-borne, infant, and animal botulism (different food, feces, and different organs). Samples that tested positive for C. botulinum by mouse bioassay concordantly tested positive by our multiplex and singleplex assays. With one exception, the multiplex and singleplex real-time PCR worked with DNA directly extracted from the suspicious specimens, without prior anaerobic culture, thus yielding results faster than the mouse bioassay. Nevertheless, the independent enrichment step by anaerobic culture is recommended for cases where low concentrations of bacteria are present.
Advancing the existing PCR-based detection of BoNT-producing clostridia, the present study describes a pentaplexed real-time PCR assay for the simultaneous detection of bont/a, bont/b, bont/e, and bont/f, including an IAC. Of technical interest is the successful combination of both MGB- and LNA-containing oligoprobes in the current pentaplex approach. The multiplex assay was found to be as sensitive and specific as the independently developed and validated singleplex real-time PCR assays for all relevant bont genes. Furthermore, the pentaplex assay allows the detection, differentiation, and quantification of all serotypes and subtypes associated with human botulism known thus far, including a novel A subtype identified recently in our group. Most importantly, the assay turned out to be very robust and rapid when analyzing food and clinical specimens from cases of botulism. Therefore, this assay will be of great diagnostic value as a rapid screening tool in surveillance studies or in outbreak situations.