Validation of shorter protocol for detection of Salmonella enterica subsp. Enterica in peanut butter samples followed by a rRNA detection system

By: Kathleen Merx1 and Jvo Siegrist2, 1 BECIT GmbH, Edisonstr. 5, 06766 Wolfen, Germany 2 Sigma-Aldrich, Industriestr. 25, 9470 Buchs Switzerland

Abstract

Food-borne pathogens Salmonella is commonly evaluated in manufacturing of peanut butter and other food products. For the HybriScan®D Salmonella Test (Product No. 55662) the ISO based enrichment method is recommended. That means sample pre-enrichment for 18 hours at 37 °C in buffered peptone water (BPW) followed by a selective enrichment step in Rappaport-Vassiliadis (RV) Broth for 24 hours at 41 °C. HybriScan®D Salmonella is a rRNA sandwich hybridisation detection system which needs at least 500 cfu/ml for the assay. This rapid molecular test system is desirable for the detection of Salmonella species like S. Enteritidis, S. Typhimurium, S. Typhi and S. Paratyphi. Results of this study on a rapid test kit demonstrate that Salmonella enterica subsp. enterica (ATTC 13311) can be detected and identified in a shorter time even in a difficult sample matrix like peanut butter.

Introduction

Peanut butter consist of about 20% carbohydrates, 25% proteins and 50% fat, Salmonella cells are just a very small component of the overall sample material and may be attached within the food matrix as single cells or clumps of cells. Normally before rapid detection methods can be used successfully, it is usually necessary to separate the target cells from the food matrix and from the background microflora. But even the HybriScan is as well a rapid molecular biological system it is practically insensitive to the sample matrix.

HybriScan®D Salmonella test is based on the detection of target molecules from the microorganisms of interest by means of specific capture and detection probes in a so-called sandwich hybridization. The hybridization reaction of the target molecules with the Biotin-labeled capture and a DIG-labeled detection probe takes place in a streptavidin coated microtiter plate (Figure 1).

sandwich-hybridization

Figure 1. Sandwich Hybridization between Target rRNA Capture and Detection Probe.

After coupling of the target molecule to the microtiter plate, an enzyme is attached in a subsequent incubation step.
After several washing steps, reaction with a color substrate gives blue coloration that changes into yellow color after the
addition of a stop solution. The yellow color enables highly sensitive photometric measurement at 450 nm (Figure 2).
Comparison is made with the standard solutions contained in the test kit.

work-flow-hybriscan

Figure 2. Work flow – HybriScan®D Salmonella.

Experimental

The matrix to be examined was Peanut Butter (9 different brand codes with 12 different code dates). The pre-enrichment time described in the protocol of the HybriScan®D Salmonella test takes 42 hours. In this experiment one target was to reduce the enrichment time to 24 hours in total. The cultivation time of the pre-enrichment peptone water culture took 18 hours; the incubation time of the selective enrichment culture in Rappaport-Vassiliades Enrichment Broth was shortened to 6 hours. Per peanut butter batch, one sample wasn't inoculated and carried as a negative control (N.C.),9 to 10 samples were inoculated with Salmonella enterica subsp. Enterica (ATTC 13311). Each Peanut Butter sample was treated as follows:

  • 25 g of Peanut Butter (except the negative controls) were inoculated with 1 to 5 cells of Salmonella enterica subsp. Enterica.
  • 225 mL buffered peptone water were added to each sample, the mixture was homogenised for 1 minute in a Stomacher and the sample were incubated for 18 hours at 37 °C.
  • After 18 hours of incubation 0.1 mL of the pre-enrichment peptone water culture were transferred to 10 mL
    Rappaport-Vassiliades Enrichment Broth. The selective main enrichment was conducted for 6 hours at 41°C.

Cell lysis and the HybriScan®D Salmonella assay were completed as described in the HybriScan®D Salmonella  test protocol. In addition, each negative control and inoculated sample were tested for Salmonella according to EN ISO 6579:2002.

Results

Evaluation of the samples was performed using the following formula as described in the HybriScanD® Salmonella assay:

Sample O.D.% = (O.D.sample ̶ O.D.N.C.) ̸ (O.D.P.C. ̶ O.D.N.C.) × 72.1%

P.C. positive control (S3)

N.C. negative control (S1)

Samples with O.D.% values under 10 are considered negative.
Samples with O.D.% values from 10 to <20 are considered questionable.
Samples with O.D.% values ≥10 are considered positive.

Of the 107 inoculated with Salmonella enterica peanut butter samples, 106 were identified as clearly contaminated with Salmonella by the use of HybriScan®D Salmonella assay. The result of 1 sample was considered questionable. All negative controls gave negative results in the HybriScan®D Salmonella assay. See Table 1– 4.

Table 1
 

Code Date Probe Average O.D. 450 nm Sample O.D.% Cells/ 10 µL
  S1 0.046   0
  S2 0.149   10.0
  S3 0.377   30.0
  S4 1.017   90.0
1624251 1-1 N.C. 0.046 0.0  
  1-2 4.060 874.2  
  1-3 3.955 851.4  
  1-4 3.970 854.6  
  1-5 3.899 839.2  
  1-6 3.891 837.4  
  1-7 3.535 760.0  
  1-8 3.791 815.8  
  1-9 3.736 803.7  
  1-10 3.816 821.1  
1874251 2-1 N.C. 0.048 0.3  
  2-2 3.748 806.4  
  2-3 3.726 801.6  
  2-4 4.072 877.0  
  2-5 3.907 840.9  
  2-6 3.965 853.5  
  2-7 3.824 822.8  
  2-8 4.060 874.2  
  2-9 4.003 861.9  
  2-10 4.032 868.2  

 

Table 2
 

Code Date Probe Average O.D. 450 nm Sample O.D.% Cells/ 10 µL
  S1 0.051   0
  S2 0.169   10.0
  S3 0.409   30.0
  S4 1.171   90.0
01574251 3-1 N.C. 0.057 1.2  
  3-2 0.608 112.1  
  3-3 1.665 324.6  
  3-4 0.289 47.9  
  3-5 0.120 13.9  
  3-6 1.146 220.2  
  3-7 0.701 130.8  
  3-8 0.251 40.2  
  3-9 0.981 187.0  
  3-10 0.359 62.0  
01334252 4-1 N.C. 0.051 0.0  
  4-2 3.737 741.4  
  4-3 3.682 730.3  
  4-4 3.927 779.6  
  4-5 3.700 733.9  
  4-6 3.780 750.1  
  4-7 3.813 756.6  
  4-8 3.915 777.1  
  4-9 3.682 730.4  
  4-10 4.091 812.6  
00864252 5-1 N.C. 0.053 0.4  
  5-2 0.232 36.4  
  5-3 0.267 43.4  
  5-4 0.740 138.7  
  5-5 0.880 166.7  
  5-6 0.736 137.8  
  5-7 0.561 102.7  
  5-8 1.967 385.3  
  5-9 2.093 410.7  
  5-10 3.585 710.7  

 

Table 3
 

Code Date Probe Average O.D. 450 nm Sample O.D.% Cells/ 10 µL
  S1 0.058   0
  S2 0.154   10.0
  S3 0.356   30.0
  S4 0.982   90.0
93294252 6-1 N.C. 0.046 -2.8  
  6-2 3.727 886.2  
  6-3 3.994 950.8  
  6-4 3.659 869.8  
  6-5 3.512 834.4  
  6-6 3.430 814.5  
  6-7 3.588 852.8  
  6-8 1.738 405.9  
  6-9 4.067 968.5  
  6-10 3.727 886.2  
01304252 7-1 N.C. 0.046 -2.9  
  7-2 3.787 900.7  
  7-3 3.575 849.5  
  7-4 3.779 898.8  
  7-5 3.875 922.0  
  7-6 4.144 987.1  
  7-7 4.067 968.5  
  7-8 3.988 949.4  
  7-9 4.135 984.9  
  7-10 4.101 976.7  
01304252 8-1 N.C. 0.053 -1.2  
  8-2 3.507 833.2  
  8-3 3.906 929.4  
  8-4 3.708 881.7  
  8-5 3.672 873.1  
  8-6 4.059 966.4  
  8-7 3.914 931.5  
  8-8 4.101 976.7  
  8-9 3.750 891.8  
  8-10 3.852 916.5  

 

Table 4
 

Code Date Probe Average O.D. 450 nm Sample O.D.% Cells/ 10 µL  
  S1 0.047   0  
  S2 0.131   10.0  
  S3 0.339   30.0  
00984252 9-1 N.C. 0.060 3.1    
  9-2 2.749 668.2    
  9-3 0.267 54.4    
  9-4 1.231 292.9    
  9-5 0.166 29.3    
  9-6 1.516 363.3    
  9-7 2.969 722.7    
  9-8 1.921 463.5    
  9-9 3.646 890.1    
00894252 10-1 N.C. 0.050 0.7    
  10-2 1.372 327.7    
  10-3 3.943 963.6    
  10-4 4.063 993.3    
  10-5 3.913 956.2    
  10-6 3.543 864.7    
  10-7 3.803 929.0    
  10-8 3.869 945.3    
  10-9 4.112 1005.4    
  10-10 3.615 882.4    
01904252 11-1 N. C. 0.049 0.5    
  11-2 3.537 863.1    
  11-3 3.874 946.6    
  11-4 3.988 974.8    
  11-5 3.979 972.4    
  11-6 4.049 989.7    
  11-7 3.881 948.2    
  11-8 3.421 834.5    
  11-9 3.984 973.7    
  11-10 3.635 887.3    
00704252 12-1 N.C. 0.051 0.9    
  12-2 2.225 538.6    
  12-3 3.100 755.0    
  12-4 2.251 545.0    
  12-5 0.434 95.7    
  12-6 1.037 244.7    
  12-7 0.340 72.5    
  12-8 0.555 125.5    
  12-9 0.293 60.7    

 

S1= Standard1 0 cells/10 µL

S2=Standard2 10.000 cells/µL

S3=Standard3 30.000 cells/10µL

S4= Standard4 90.000 cells/10µL

N.C.= Negative control: sample was not inoculated with Salmonella spp.

Materials

     
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