AIM
Diagnosis of MAP infection is a challenge due to the chronic nature of the disease and the occurrence of four stages. Most animals shed the bacteria long before the appearance of clinical signs and these shedders spread the infection to other animals by contaminating the environment. Thus, it is important to diagnose the presence of the bacteria long before the clinical signs appear. Real-time PCR method such as Bio-T kit® Mycobacterium avium paratuberculosis enables sensitive amplification and quantification of MAP target. However, the process of analysis can be time-consuming and labor-intensive, especially in the context of quantitative analysis. For this reason, BioSellal has evaluated a robust qPCR solution that is portable, reliable, and able to automatically analyze quantitative results.
METHODS
Faeces Extraction
Faeces field samples selected from different herds in Creuse (France) had been previously characterized by PCR. Sample Pretreatment and DNA extraction were performed as detailed in the extraction manual of the Bio-T kit® Mycobacterium avium paratuberculosis using BioExtract® Superball® (BioSellal, France).
MRSI Sample
Reference Material Set at the Interpretation threshold (MRSI, Cat. N° MRSI-MAP-001) is constituted by a negative faeces sample for MAP supplemented at 104 Equivalent Genome (EG) / g with a titrated bacterial suspension of MAP (SB-MAP-001). This level is used as a threshold to identify high shedders (bacterial load > 104 EG/g) and low shedders. This MRSI sample has been extracted as a field sample.
DNA standards
Synthetic MAP DNA was serially 10-fold diluted from 5.103 to 0.5 EG per reaction in Nuclease-Free Water corresponding to 1.57x107 to 1.57x103 EG/g of faeces.
Reaction Set Up
5μl of DNA was mixed with 15 μl of Bio-T kit® Mycobacterium avium paratuberculosis’ Master Mix in a plate for 7500 Real-time PCR System and in Mic tube for Mic qPCR Cycler. This mixing step was performed using the Myra Liquid Handling system.
Cycling Parameters
All samples and no-template controls were amplified on an Applied Biosystems 7500 Fast Real-Time PCR System in standard mode and on BMS Mic qPCR Cycler in fast mode. Cycling parameters of 7500 Fast were set up according to the Bio-T kit® Mycobacterium avium paratuberculosis’s instructions. For the Mic qPCR Cycler program, please contact BioSellal.
RESULTS
To evaluate the possibility to use Bio-T kit® Mycobacterium avium paratuberculosis on Mic qPCR Cycler field samples were analyzed and compared with 7500 Fast Real-Time PCR System.
Figure 1: Curve' shape comparison between 7500 Fast Real-Time PCR System and Mic qPCR Cycler with Bio-T kit® Mycobacterium avium paratuberculosis
=> The shape of the amplification curves of Bio-T kit® Mycobacterium avium paratuberculosis for MAP and exogenous IPC are equivalent between 7500 Fast Real-Time PCR System and Mic qPCR Cycler.
To validate quantification parameters, serially diluted synthetic MAP DNA standard and MRSI samples were analyzed and compared with the 7500 Fast Real-Time PCR System.
Figure 2: Curve and efficiency comparison between 7500 Fast Real-Time PCR System and Mic qPCR Cycler with Bio-T kit® Mycobacterium avium paratuberculosis
=> The shape of the amplification curves for MAP target on DNA standards and MRSI are equivalent between 7500 Fast Real-Time PCR System and Mic qPCR Cycler.
Table 1: Ct values of DNA standard curve and MRSI sample: evaluation of quantification parameters between 7500 Fast Real-Time PCR System and Mic qPCR Cycler with Bio-T kit® Mycobacterium avium paratuberculosis
=> Efficiency and quantification parameters for MAP target on DNA standards and MRSI are equivalent between 7500 Fast Real-Time PCR System and Mic qPCR cycler.
Then, the Ct values of previously stated field samples were compared between 7500 Fast Real-Time PCR System and Mic qPCR Cycler.
Table 2: Ct values comparison for field samples between 7500 Fast Real-Time PCR System and Mic qPCR Cycler with Bio-T kit® Mycobacterium avium paratuberculosis
* Sample with a bacterial load below the limit of quantification
=> Ct values for MAP target on field samples are equivalent between 7500 Fast Real-Time PCR System and Mic qPCR Cycler except one field sample detected on 7500 Fast Real-Time PCR System that was not detected on MIC PCR cycler. Nevertheless, this sample is a very weak positive sample with a viral load below the limit of detection and quantification.
To make easier and faster the analysis of absolute quantification or relative quantification of MAP, BioSellal has developed an automated template on MIC software.
Figure 3: Automatized analysis on MIC software: relative quantification toward MRSI sample
=> Using a ready-to-use template on the very intuitive MIC software, relative quantification of MAP toward MRSI has been automatized. The status of the sample is indicated with a user-friendly color code and a relative quantification value is indicated for each sample.
Figure 4: Automatized analysis on MIC software: absolute quantification of MAP bacterial load
=> Using a ready-to-use template on the very intuitive MIC software, absolute quantification of MAP has been automatized. The absolute quantification value is indicated for each sample.
CONCLUSIONS
BioSellal has evaluated a new sensitive and rapid method using the Bio-T kit® Mycobacterium avium paratuberculosis on Mic qPCR Cycler. This method can be used for absolute or relative quantification of MAP from faeces samples to rapidly identify MAP making it easier to screen animals’ samples and detect MAP earlier before symptoms appear. The Mic qPCR Cycler can test up to 48 samples at once in just 38 minutes. This run gave Ct curves and PCR efficiency identical to classical real-time PCR machines that are much bulkier. The performances are equivalent to a gain in speed and portability. Moreover, it eliminates the need for calibration, annual service, and preventative maintenance. Thanks to very intuitive software, automatized analysis has been developed by Biosellal either for relative quantification toward MRSI samples or absolute quantification. All these data allow an easier and faster MAP diagnosis.
ORDERING INFORMATION
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