Authors: John Bodner, PhD – Nevin Perera, PhD – Holly Urquhart – Erin Caruthers, PhD |
On-site pathogen detection at food production facilities is now possible due to technology advances, which allow for faster recognition and remediation. Immunoassays are an acknowledged method for sensitive, specific and rapid food pathogen testing. CERTUS has incorporated a Raman-based immunoassay into their automated detection system, which is made safe by performing the enrichment and measurement inside a detection tube that is never opened after enrichment begins.
This study aims to characterize a novel detection format (Surface Enhanced Raman Spectroscopy immunoassay) developed for the detection of environmental Listeria species in the presence of a large foam collection swab. The swab is pre-wetted with a neutralizing buffer and there is no sample manipulation post-collection. The assay is run on the CERTUS BIO-Lock™ System in a single enrichment media that allows for the simultaneous growth and detection of the Listeria bacterial pathogen in a closed system using the CERTUS EL Detection Kit.
Materials and Methods
For the inclusivity and exclusivity study evaluations, 42 Listeria serotypes and 44 non-Listeria strains were cultured in 15 mL CERTUS proprietary enrichment media at inoculation level ranges of 10 – 100 for Listeria and 103 – 106 cfu for non-Listeria respectively. Enrichment then commenced for 24 h at 30 ± 1°C.
For Listeria species growth profile evaluation, serotypes were cultured in 15 mL CERTUS proprietary media at inoculation level ranges of 1 – 104 cfu. Enrichment then commenced for 24 h at 30 ± 1°C.
For bio-burden study evaluations, Listeria species at inoculation level ranges of 5 – 20 cfu and environmentally isolated Gram Positive bacterial microflora at a low bio-burden 103 cfu or a high bio-burden 104 cfu were co-inoculated. The competitive mixes were cultured in 15 mL CERTUS proprietary enrichment media for 24 h at 30 ± 1°C.
For environmental surface dry-down studies, 4 different surfaces (stainless steel, plastic, ceramic and sealed concrete) were evaluated at low and high Listeria inoculation levels. Different Listeria species were inoculated at low 103 cfu or high 104 cfu levels onto surface matrices, except for stainless steel where the Listeria species was co-inoculated with a 10-fold excess of a competing Enterococcus faecalis at both low and high levels. Surfaces were then dried for 16 – 24 h at room temperature (24 ± 2°C) prior to sampling. Surfaces were sampled with the collection device using horizontal and vertical sweeping patterns, and then cultured in 15 mL CERTUS proprietary enrichment media for 24 h at 30 ± 1°C.
To estimate how soon a presumptive positive could be called relative to the Listeria level present in the assay, a series of serial dilutions were run with L. monocytogenes 4b ATCC19115 (Figure1). Signal is monitored continuously over time. The time to result is calculated through a proprietary algorithm that monitors deviation from a straight line. The observed trend was as expected in that at a high level of Listeria (104 cfu – present in biofilms) the curve inflection correlates to Time to Result (TTR) is around 8 to 9 hours while at a fractional level of Listeria (3 cfu) the inflection occurs later and is around 17 to 18 h.
Figure 2 shows the growth profiles of 7 serotypes of Listeria monocytogenes 6 out of the 7 samples were at fractional levels and all showed a TTR of < 20 h. L. monocytogenes 1/2b that was 1 cfu returned a TTR of 18 h.
Figure 3 shows the growth profiles for six classical Listeria species. The inoculation level ranges from 3 to 19 cfu and all species were detected with a TTR of < 20 h.
Table 1 shows the recovery across multiple Listeria species in the presence of a low and high bio-burden. Environmentally isolated Gram Positive bacterial microflora isolated from washes of produce were subsequently enumerated and the samples were inoculated into the assay to achieve a low bio-burden 103 or a high bio-burden 104 competitive mix. In this competition assay the TTR’s were extended relative to what was observed in Figures 1 and 2, however, the etection of Listeria was comfortably obtained in under 24 h.
Recovery of dried down Listeria on 4 different surfaces was studied. As indicated both low and high inoculation levels were tested plus for stainless steel Enterococcus faecalis was co-inoculated on the surface. Table 2 shows the recovery results. As expected there was a range of TTR’s observed given the inherent variation in the replicates of the dried down samples. Again, in this recovery study the TTR’s were extended, but again recoveries were comfortably obtained under 24 h.
Inclusivity and exclusivity studies were run to assess the overall accuracy of the assay system. A 42-member Listeria inclusivity panel containing type strains and naturally isolated wild types was run. The exclusivity panel was made up of 44 non-Listeria members. Table 3 and 4 shows the tabulated results.
Table 3: Inclusivity Samples
Table 4: Exclusivity Samples
Table 5 shows the positive and negative agreement between the CERTUS EL assay and confirmation assay either by BAM or plating on selective chromogenic agar. The total number samples tested was 663. It was observed to be 473 positive agreements and 176 negative agreements with 11 negative deviations and 3 positive deviations. These results lead to a calculated assay sensitivity and specificity of 98%.
Table 6 shows a direct correlation on a total of 74 samples between the CERTUS Listeria test method and BAM Ch 10 reference method on four environmental surfaces. There were 3 positive and 3 negative deviations observed.
Table 6: Comparison of CERTUS Listeria test method vs. BAM Ch 10 reference method on four environmental surfaces
To estimate the Limit of Detection / Analytical sensitivity of the CERTUS EL assay a L. monocytogenes 4b ATCC19115 was cultured from a starting inoculation level of 14 cfu. At specified time points, enumerations were carried out using a Miles & Misera methodology to calculate the overall cell density and identify the level at the curve inflection which correlates with the assays’ analytical sensitivity. This was found to be around 5×105 (Figure 4).
The CERTUS EL assay successfully recovers Listeria species from multiple environmental surfaces (stainless steel, plastic, ceramic and sealed concrete) analyzed either in the presence or absence of competing bacterial microflora at varying bio-burden levels within a specified 24 h timeframe.
The CERTUS EL assay has the capability of detecting all ‘classical’ Listeria species and multiple serotypes of Listeria monocytogenes in a time dependent manner that inversely correlates with relative starting Listeria levels – the higher the starting Listeria cell level the earlier the curve inflection and Time to Result.
The results of the inclusivity and exclusivity evaluation confirms the high specificity and selectivity of the CERTUS EL assay to Listeria species with an overall 98% calculated specificity level.
Sensitivity testing between the CERTUS EL assay test method and BAM Ch 10 reference method showed a high correlation between positive and negative agreements from a 663 sample pool resulting in an overall 98% calculated sensitivity level. There were 3 positive and 3 negative deviations observed from 74 environmental surface samples tested across the four matrices.
Analysis of a Listeria monocytogenes 4b growth profile and determination of cell density at varying time points calculated the CERTUS EL assay Limit of Detection to be around 5×105 – 1×106 cfu/mL.
This new pathogen detection method combines real-time monitoring, selective enrichment, sensitive and specific detection in a single tube; giving food producers a safe, simple and accurate method of detecting environmental pathogens on-site.