Molecular solutions to improve sensitivity and specificity while reducing process time.
Food Safety Testing Molecular solutions to improve sensitivity and specificity while reducing time-to-results
The promotion of a high level of food safety is a major priority worldwide. Detecting the presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, the correct animal species in processed meat and the correct labelling in foods suitable for vegetarians or those with allergies are critical to quality assessment programmes within the food industry. Conventional microbiological methods based on culturing techniques are slow and can be insensitive. A growing alternative approach that is faster, more selective, capable of multiplexing and highly sensitive is molecular testing. Meridian’s optimized qPCR and RT-qPCR mixes offer ideal solutions for molecular food testing assays. Specifically, Meridian’s Air-Dryable mixes are a new practical alternative for assays using crude samples. The mixes are inhibitor-tolerant, exhibit high sensitivity across a wide dynamic range, and can be air-dried to create ambient-temperature stable assays. Assays using these mixes have the same performance whether they are used in a wet or dry format, with the dried assays having the added benefit of simpler cold chain logistics and extended shelf-life, both features that improve the cost of food safety management.
Molecular Food Safety Tests
There are 31 identified foodborne pathogens in the United State and it is estimated that viruses are the primary cause of illnesses whereas bacteria are the primary cause of hospitalizations and deaths ( Scallan, E., et al. (2011)). The common foodborne pathogens which are responsible for most of the foodborne disease outbreaks are Listeria monocytogenes, Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enterica, Bacillus cereus , Vibrio spp., Campylobacter jejuni, Clostridium perfringens , and Shiga toxin-producing Escherichia coli (STEC) (Oliver, S. P., et al. (2005)). Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. Foodborne Pathog. Dis. 2, 115–129). Reducing the time needed to carry out pathogen detection tests has been a primary goal for food microbiologists for several decades. The advantages to PCR-based testing methods are that they are much more sensitive than culturing and they do not require enrichment of the organism in order to detect very low levels. In addition, testing can be automated and performed on crude samples to give highly reproducible results. Pathogen Detection to Prevent Foodborne Illness
Air-dryable mix demonstrates high sensitivity of pathogens across a wide dynamic range (10 6 to 10 cfu) in a wet or dry format
A) Staphylococcus aureus amplification plot
B) Listeria monocytogenes amplification plot
Staphylococcus aureus standard curve
Listeria monocytogenes standard curve
Figure 1 . Using Air-Dryable ™ qPCR Mix (MDX082), amplification of 16s genes from, A/ Staphylococcus aureus and B/ Listeria monocytogenes was compared in a 10-fold dilution series before ( blue ) and after ( red ) air-drying. The results illustrated that as low as 10 cfu/rxn could be detected and that the reaction efficiencies were also maintained before and after drying at 98% and 100% respectively.
Air-dryable mix exhibits high inhibitor tolerance and provides sensitive results using crude samples
Figure 2 . Amplification of 10-fold serial dilution of Hepatitis A RNA using air-dried Air-Dryable ™ 1-Step RT-qPCR Mix (MDX095) in presence of 0.8% seafood extract. The results
illustrated the sensitivity of the Air-Dryable ™ 1-Step RT-qPCR Mix, being able to detect very low levels of Hepatitis A, even in a crude sample.
Food Authenticity Testing to Protect Consumer Interests and Public Health
Authenticity testing is utilized to prove that the content of food products are authentic and the way they are presented is correct and accurate. Government regulations are in place to verify the authenticity and origins of a product, including those that are sourced internationally. Intentional adulteration or counterfeiting is considered food fraud and can result in billions in economic loss and long-term damage to brand reputation. It can also pose a serious threat to public health. Common testing for food fraud includes GMO screening and meat or fish species identification.
Air-dryable mix exhibits high specificity for detecting low level contaminants in complex food samples
Figure 3 . Detection of Bovine mitochondrial ATP Synthase DNA in extracts of beef meat (black), pork meat ( pink ) and pork meat contaminated with 0.4% beef ( red ) using air-dried Air-Dryable ™ qPCR Mix. The results illustrated specificity of the Air-Dryable ™ qPCR Mix, being able to distinguish the pork meat contaminating the beef product.
Figure 4 . A 10-fold serial dilution of Bovine mitochondrial ATP Synthase DNA was amplified over 4 orders of magnitude using the Air-Dryable ™ qPCR Mix. The results illustrated sensitivity and reproducibility of the Air-Dryable ™ qPCR Mix, being able to detection down to 1 genome equivalent ( blue ) with very good correlation coefficient and reaction efficiency.
Benefits to Room-Temperature Stable Assays Room-temperature stable assays are becoming increasingly popular with test manufacturers looking to simplify assay handling and processing. The main benefits are eliminating the need for cold storage, increasing the shelf life of products at ambient temperatures and enabling ‘one tube’ assay protocols. Overall, ambient-temperature stable assays help reduce the cost of shipping and storage and increase the life of the product. Air-Dryable RT-qPCR and qPCR mixes are easy to use and ideal for developing ambient temperature, multiplex molecular assays. They are glycerol-free and contain Taq polymerase, reaction buffer, dNTPs, MgCl 2 and air-dry compatible excipients. In order to produce room temperature air-dried qPCR reagents, assay specific primers and probes can be added to Air-Dryable qPCR Mix for subsequent air-drying.
Stable shelf-life for up to 12 months
Figure 5 . Air-Dryable ™ qPCR Mix was air-dried, and the stability was tested in an accelerated stability study. The air-dried mix was incubated at 37°C for 8 weeks ( black ) and tested against a freshly prepared mix ( red ) by qPCR assay on 100-fold template dilutions. Results suggest that the air-dried mix is active following accelerated stability tests with projected 12 months stability at ambient temperature.
Reference | 37°C
Optimized Master Mixes
MDX030 Low DNA qPCR Mix Designed for multiplex assays detecting microbial or fungal DNA. Incorporates a heat-activated DNA polymerase with low residual DNA content. MDX020 Fast qPCR Mix Ideal for multiplex assays requiring sensitive detection of DNA targets in inhibitor-rich samples. Contains an antibody-mediated hot-start polymerase. MDX032 Fast 1-Step RT-qPCR Mix Ideal for multiplex assays requiring sensitive detection of DNA and RNA targets. Contains an antibody-mediated hot-start polymerase, MMLV-RT and separate RNase inhibitor. MDX013 Inhibitor-Tolerant qPCR Mix Designed for amplification direct from crude lysates or inhibitor-rich samples such as urine, cerebral spinal fluid (CSF), blood as well as plants. Contains an antibody-mediated hot-start polymerase. MDX016 Inhibitor-Tolerant RT-qPCR Mix All-in-one, 4x master mix designed for RT-qPCR from crude samples such as sputum, stool and saliva. No sample extraction required.
MDX082 Air-Dryable ™ qPCR Mix, 4x Ready-to-use qPCR mix ideal for oven or air-drying technologies. MDX095 Air-Dryable ™ RT-qPCR Mix, 4x Ready-to-use RT-qPCR Mix ideal for oven or air-drying technologies. MDX021 Lyo-Ready ™ qPCR Mix Ready-to-use, glycerol-free qPCR master mix formulated with a specialized blend of excipients for lyophilization into beads or pellets. MDX024 Lyo-Ready ™ 1-Step RT-qPCR Mix Ready-to-use, glycerol-free RT-qPCR master mix formulated with a specialized blend of excipients for lyophiliization into beads or pellets. MDX062 Lyo-Ready ™ 1-Step RT-qPCR Virus Mix Ready-to-use, glycerol-free RT-qPCR master mix formulated with a specialized blend of excipients and highly suited for amplification of RNA viruses with a high secondary structure. MDX097 Lyo-Ready ™ LAMP Mix Ready-to-use, glycerol-free RT-qPCR master mix formulated with a specialized blend of excipients for lyophilization into beads or pellets.
Molecular Reagents continued..
TAQ POLYMERASES MDX011
ISOTHERMAL POLYERMASES MDX012 Bst DNA Polymerase
Glycerol-Free Taq HS 50 U/μL Lyophlization-compatible, high concentration (50 U/μL), glycerol free DNA enzyme for automated high-throughput testing (provided as separate antibody, enzyme and dilution buffer). MDX015 Aptamer Taq HS (Glycerol-Free) A high concentration, lyophilization-compatible Taq DNA polymerase containing a DNA aptamer which binds reversibly to the polymerase. Suitable for developing highly specific, high-throughput assays. MDX009 Low DNA Taq HS 5 U/μL Heat-activated, thermostable DNA polymerase with low residual DNA content. Ideal for multiplex assays involving amplification of bacterial and fungal DNA. MDX010 Low DNA Taq HS 10 U/μL Heat-activated, thermostable DNA polymerase with low residual DNA content. Ideal for multiplex assays involving amplification of bacterial and fungal DNA. MDX001 Taq DNA Polymerase DNA polymerase with optimized buffer system for fast PCR reactions across a range of templates. MDX008 Taq DNA HS Polymerase Antibody-mediated hot-start DNA polymerase that is ideal for developing multiplex assays requiring sensitive detection of DNA targets in inhibitor-rich samples.
Stable, heat resistant DNA polymerase optimized for LAMP and contains 5´ > 3´ DNA polymerase activity and strong strand displacement activity. MDX018 High Conc. Glycerol-Free Bst Stable, heat resistant DNA polymerase optimized for LAMP and lyophilization-compatible with a concentration of 100 U/µL. MDX019 Inhibitor-Tolerant Bst Buffer Optimized buffer for MDX012 and MDX018 that increases the amplification speed, sensitivity, and tolerance to salt and other inhibitors found in clinical samples and transport media. REVERSE TRANSCRIPTASES MDX044 MMLV-RT MMLV-RT suitable for generating first strand cDNA of up to 9 kb. Suitable for multiplex reactions. MDX043 RNase-Tolerant MMLV-RT Mixture of MMLV-RT and RNase inhibitor that maintains activity up 50° C. Suitable for detecting very low levels of RNA in inhibitor-rich samples.
MDX004 Tissue Extract-PCR Buffers Lysis and neutralization buffers optimized for use with Taq HS DNA Polymerase (MDX008) to perform PCR direct from crude lysate. MDX033 Fast qPCR Buffer, 4x Optimized for use with Taq HS DNA Polymerase (MDX008) . MDX034 1-Step RT-qPCR Buffer, 4x Optimized for use with Taq HS DNA Polymerase (MDX008) and RNase-Tolerant MMLV-RT (MDX043) . MDX056 RNase Inhibitor Inhibits a broad spectrum of eukaryotic RNases, including RNases A, B and C to control for contaminants in RT-PCR assays. MDX014 Taq HS Antibody A mix of anti-Taq antibodies designed to inhibit Taq DNA polymerase activity at room temperature. For use in hot-start PCR.
Quick Molecular Product Selection Chart
Low DNA qPCR Mix
Fast qPCR Mix
Fast 1-Step RT-qPCR Mix
Inhibitor-Tolerant qPCR Mix
Air-Dryable TM qPCR Mix, 4x
Air-Dryable TM RT-qPCR Mix, 4x
Inhibitor-Tolerant RT-qPCR Mix
Lyo-Ready TM qPCR Mix
Lyo-Ready TM 1-Step RT-qPCR Mix
Lyo-Ready TM 1-Step RT-qPCR Virus Mix
Lyo-Ready TM LAMP Mix
Glycerol-Free Taq HS
Aptamer Taq HS (Glycrol-Free)
Low DNA Taq HS
MDX009 & MDX010
Taq DNA Polymerase
Taq DNA HS Polymerase
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