Wed, 06/06/2018 - 14:27

Molecular Biology

Molecular biological analyses are best suited for identifying substances harmful to health in food and animal feed, such as Salmonella, Listeria, viruses, allergens, genetically modified organisms (GMOs) and residues. We can also use these tests to determine the gender of beef or pork.

We apply highly sensitive methods using real-time PCR, and in many cases rapid detection is possible as well. In addition, we use ELISA procedures to verify proteins in food products.

Rapid Detection of Pathogenic Germs

Article 64 of the German Food and Feed Code (LFGB) includes an official list of examination methods to detect pathogenic microorganisms like Salmonella in food products. However, the time associated with these methods is up to five days, which is too lengthy for industry and producers. The highly sensitive and specific real-time PCR method offers rapid detection for faster approval than five days for further processing and delivery, reduced storage time and ongoing monitoring during production with the opportunity of early intervention.

Salmonella Rapid Detection Using Real-Time PCR

Salmonellosis is a common infection spread through food that occurs worldwide. It generally appears as an acute intestinal inflammation with suddenly occurring diarrhea, headaches and stomach aches as well as nausea, often accompanied by vomiting and a slight fever. The illnesses can occur as single cases, case accumulations (e.g. within families) or larger outbreaks with many people affected. An infection occurs by oral intake of the pathogen.

Our rapid method of detecting Salmonella (using real-time PCR) can provide results in just 12 to 24 hours, depending on the sample matrix.

Rapid Analysis: Report in 12 hours

The 12-hour processing time is available for raw, unseasoned fresh meat. When samples are received early in the day, the reports can be transferred on the same day.

Rapid Analysis: Report in 24 hours

After an enrichment time of 16 hours for food products (including raw materials, interim products and finished products) and animal feed, you generally receive your reports no more than 24 hours after receipt of the samples.

Listeria Rapid Detection Using Real-Time PCR

Listeria (especially L. monocytogenes) can cause a disease called listeriosis, in particular in people with a weakened immune system. Compared to other foodborne illnesses, the numbers of severe cases and associated deaths of Listeria are comparatively high. Listeria is not only found in animal food products such as poultry, meat, fish, milk and milk products, but also in plant-based food products such as precut salads. Listeria can even grow in cold, refrigerator temperatures.

As a food testing laboratory with expertise in meat and all other animal products, we offer highly sensitive real-time PCR rapid detection of Listeria, in addition to the classic verification and counting methods. We use this method for detecting Listeria species as well as Listeria monocytogenes, thereby increasing the speed of processing raw products and the approval of finished products.

Rapid Analysis: Report in 24 Hours

The reports for real-time PCR detection of Listeria spp. and/or Listeria monocytogenes are generally available next day (after approximately 24 hours).

STEC: Rapid Detection Using Real-Time PCR

Enterohemorrhagic Escherichia coli (EHEC) are intestinal bacteria with the characteristic of forming Shiga toxins (Stx) and are therefore also referred to as Shiga toxin or verotoxin producing E. coli (STEC or VTEC). STECs are defined via the presence of the Shiga toxin gene (Stx1 or Stx2) in the genome. The eae-gene is another virulence marker. STECs are highly pathogenic for humans and can cause hemorrhagic colitis and hemolytic-uraemic syndrome (HUS). The most frequently isolated EHEC serogroup is O157. However, in connection with EHEC diseases in humans, new serogroups or serovars continue to be detected.

Rapid Analytics: Report in 24 hours

The reports for real-time PCR detection of STEC (screening for stx1 / stx2 genes) are generally available the next day (after approximately 24 hours).

For positive STEC screening results, we can perform examinations for the serogroups O26, O45, O103, O111, O121 and O145 and for E. coli O157:H7 (which are most often associated with severe illnesses).

Virus Detection

Viruses relevant to food products include adenoviruses, rotaviruses, diverse hepatitis viruses and the noroviruses. The latter play the most significant role as one of the most frequent causes of viral gastroenteritis.  The direct transferability from human to human is the most significant cause for the high number of norovirus infections. The transfer occurs fecal-orally (for example through hand contact with contaminated surfaces) or via oral intake of virus-containing drops. Infections can, however, also originate from contaminated meals (such as salads, crabs and mussels) or drinks (contaminated water). Viruses, unlike bacteria, can,  not multiply outside of a living organism. However, they are extremely stable in food products and even remain infectious with refrigeration and deep freezing. The infectivity is very high, which explains the rapid spread of viruses within nursing homes, hospitals and community facilities.

Norovirus and Hepatitis A Virus Detection Using Real-Time PCR

We detect norovirus and hepatitis A virus through  multiplex real-time RT-PCR for direct qualitative verification.

Allergen Verification

A number of food products and additives can trigger allergies. Even in the smallest quantities they can lead to considerable, or even life-threatening, immune reactions in people with allergies or sensitivities. To protect this population, the German Food Product Labelling Ordinance (LMKV) defines 14 common allergens that must be included on food product labels.

Allergen labeling has been required at the European level via EU Directive No. 1169/2011 (LMIV) since late 2011, and the same 14 allergens must also be highlighted on the packaging, for example using a font or background color, and also be marked in “loose goods.”

The allergens listed in Annex II of EU Directive1169/2011 are:

  • Grain containing gluten as well as products manufactured with this grain
  • Crustaceans and products containing crustaceans
  • Eggs and products containing eggs
  • Fish and products containing fish
  • Peanuts and products containing peanuts
  • Soybeans and products containing soy
  • Milk and milk products (including lactose)
  • Nuts (almonds, hazelnuts, walnuts, cashews, pecans, Brazil nuts, pistachios, macadamia nuts or Queensland nuts) as well as products containing nuts
  • Celery and its derived products
  • Mustard and its derived products
  • Sesame seeds and its derived products
  • Sulfur dioxide and sulfites
  • Lupins and products containing lupins
  • Mollusks and products containing mollusks

We detect allergens in food products via DNA testing using real-time PCR methods or ELISA procedures, which detect proteins in food products. The methods are specific and sensitive, so that even very small traces of allergens can be detected.

For lactose, sulfur dioxide and sulfites, we use chemical detection techniques.

Allergen Detection Using Real-Time PCR

We offer DNA-based qualitative verification using real-time PCR for the following allergens:

  • Celery
  • Mustard
  • Sesame
  • Crustaceans
  • Fish
  • Peanuts
  • Soy
  • Nuts (almonds, hazelnuts, walnuts and pistachios)
  • Lupins
  • Mollusks

If any of these allergens are detected via qualitative real-time PCR, we can add a quantitative real-time PCR analysis to get a volume estimate for the following allergens:

  • Celery
  • Mustard
  • Soy

Allergens Detection Using ELISA

We detect the following allergens using ELISA procedures:

  • Gluten (R5 Mendez antibodies)
  • Milk
  • Chicken protein
Species Identification

Potential falsifications in products with non-declared animal species can be detected by examining the DNA of different types of animals. We perform this analysis using real-time PCR which delivers reliable results even for complex and highly processed products: Depending on the sample matrix and degree of processing, even 0.1 to 0.01 percent of a species can be detected. The procedure is also very well suited for screening for animal ingredients in vegan or vegetarian products as well as for HALAL certification. Using ELISA technology, we can detect proteins in the samples and verify positive PCR findings.

Species Detection  Using Real-Time PCR

We use real-time PCR to test for the following types of animals:

  • Horse
  • Donkey
  • Pig
  • Cattle
  • Chicken
  • Turkey
  • Duck
  • Sheep
  • Goat
  • Water buffalo
  • Other species on request

Upon request, we can also verify positive real-time PCR findings using ELISA technology to detect proteins in the samples. In addition, a quantification using real-time PCR is possible for cattle, pig and horse content, depending on the sample matrix. The relative quantitative determination of the DNA portion of the target species is relative to the total animal DNA portion in meat products.Auf Wunsch können positive real-time PCR-Befunde außerdem mittels Elisa-Technik abgesichert werden. Dieses Verfahren dient dem Aufspüren von Proteinen in den Proben. Außerdem ist für die Tierarten Rind, Schwein und Pferd in Abhängigkeit von der Probenmatrix eine Quantifizierung mittels real-time PCR möglich. Die relative quantitative Bestimmung des DNA-Anteils der gesuchten Tierart erfolgt relativ zum Gesamttier-DNA-Anteil in Fleischwaren.

Gender Determination for Beef and Pork

We perform gender determination for beef and pork using animal-specific y chromosome sections. Real-time PCR analysis delivers certain insights regarding the gender of the examined species even when additional species are present (such as inmixed ground meat). The material for analysis is generally taken from the core of the sample. This eliminates obtaining false results due to contact of the sample with the material from other animals (for example during the slaughtering or cutting process).

Gender Determination of Pork Using Real-Time PCR

A majority of consumers avoid the meat of uncastrated male pigs due to a somewhat unpleasant odor and taste. These specific characteristics of boar meat are caused mainly by the male steroidal sex hormone androstenone.  In the past, the creation of the distinctive odor was completely prevented by castrating male piglets.

Because real-time PCR can duplicate a specific section of the y chromosome of the male pig, correct gender assignment is possible even if the animal was castrated. Since we detect the presence of the y chromosome itself and not specific substances only found in male pigs, the time of the castration does not change the test result.

Gender Determination of Beef Using Real-Time PCR

With the help of real-time PCR, we can reliably determine the gender of the animal in beef and beef products. With this method a specific section of the cattle’s y chromosome is duplicated. The primary goal of the examination is claim verification (e.g. “from a young bull”).

Risk Materials Detection

The EU Directive 999/2001 sets measures for containing zoonosis, a disease transferred from animals to humans, e.g. Ebola virus disease and salmonellosis. These measures include a feed ban of bone meal to ruminants, the removal and destruction of specified risk materials (nerve-containing tissue such as brain, spinal cord, etc.) from carcasses, and intense, diagnostic monitoring of the slaughtered ruminants prior to release into the food chain.

Central Nervous System Disease Detection Using ELISA

We detect nerve tissue, that is specified as risk material, in meat products. Using ELISA procedures, we can examine raw meat, swab samples obtained from surfaces at the slaughterhouse and processed meat products (e.g. sausage).

Genetically Modified Organism (GMO) Detection

In the EU, food and animal feed with GMO content of 0.9 percent or higher must be labeled as containing GMOs (via Regulations (EC) No. 1139/98 and 1830/2003).

Accidental or technically unavoidable portions of approved GMO content below 0.9 percent are excluded from labeling requirements. In many regions, there are either 0 percent or very low tolerance limits for non-approved GMOs in food and animal feed. If a GMO is not approved in a specific country, but is being cultivated in another country, problems can occur in the marketability of products due to cross-contamination with GMO-free materials.

The EC Genetic Technology Implementation Law (EGGenTDurchfG) regulates labeling of food products that were manufactured without the “use of genetic engineering procedures.” The term “ohne Gentechnik” (GMO-free) is the only one approved for these food products. Trace contamination in food products and ingredients is not allowed, with contaminations above the verification limit of 0.1 percent being excluded. The Association for Food without Genetic Engineering (Verband Lebensmittel ohne Gentechnik or VLOG) issues licenses for the uniform seal "ohne Gentechnik" for food products and the seal "VLOG geprüft" (tested by VLOG) for animal feed.

We detect genetically modified organisms via DNA testing using real-time PCR, generally following this process:

  1. Screening examination
  2. Type identifications
  3. Quantification of the identified type

Screening for p35S, Tnos and CTP2 - CP4 EPSPS Using Real-Time PCR

Because the number of genetically modified organisms continues to grow, a reliable and broad spectrum screening is becoming increasingly important. Using real-time PCR analysis, we can detect three DNA sequences that frequently occur in genetically modified plants. Since most transgenic plants contain at least one of these three elements in their genome, the multiplex PCR technique is a reliable method for detecting genetically modified plant components.

These three elements are:

  • 35S- Promoter of the cauliflower mosaic virus = p35S
  • Terminator from Agrobacterium tumefaciens = Tnos
  • Transition from CTP2 to CP4 EPSPS from Agrobacterium tumefaciens, ssp. strain 4 = CTP2 - CP4EPSPS

Qualitative Detection of Specific GMO Varieties Using Real-Time PCR

We offer diverse real-time PCR detection for identifying GMO soy, GMO corn and other GMO plants. The identification is generally performed following an unusual finding in a previous screening.

We use real-time PCR for detecting these genetically modified soy varieties:

  • RoundupReady™ GTS 40-3-2
  • RoundupReady2 Yield™ MON-89788-1
  • LibertyLink™ A2704-12
  • LibertyLink™ A5547-127
  • DP305423-1 Soy
  • CV127-9 Soy
  • MON-87701-2

We use real-time PCR for detecting these genetically modified corn varieties:

  • Bt11
  • Bt176
  • T25
  • GA2
  • TC1507
  • MON810
  • MON88017

Upon request, we can perform various other GMO verifications.

GMO Quantification Using Real-Time PCR

Food and animal feed products with 0.9 percent or higher GMO content must be labeled according to Regulation (EC) No. 1830/2003. Our quantitative GMO verifications can help  determine whether the GMO content of a product lies above or under the legally prescribed threshold. We use quantitative real-time PCR to verify the GMO volume share of a food product, and thereby also the regularity of its declaration.

Residue Detection

Residues are remnants of substances that were used during the production of food products (e.g. animal medications).

Since such residues can be hazardous to human health at specific concentrations, maximum residue limits (MRLs) were set in many countries to protect consumers. Adherence to these limits is strictly controlled.

Antibiotics Detection Using ELISA

The use of antibiotics as animal medication is unavoidable, but it can cause antibiotic residues to enter the human food chain (for example through application errors) and become a health risk in food products such as milk, eggs or meat. Therefore legislation has placed numerous restrictions on the use of animal medications, from setting tolerable maximum levels for various animal varieties to use restrictions to bans.

Using ELISA procedures, we screen for the following antibiotics:

  • ß-Lactams
  • Bacitracin
  • Tetracycline

Hormone Detection Using ELISA

Ractopamine belongs to the group of ß-agonists. While these are suitable as fattening aids as part of animal production, they are not approved by the European Union.

We can detect ractopamine using ELISA technology.

Product Adulteration Detection

Products are often adulterated with additives, mostly for economic reasons. The consequence is a clear reduction in quality.

To detect adulteration in durum wheat (durum wheat, semolina), we offer a rapid membrane test.