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rRNA Methods Promise Faster Listeria Testing

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A person cleaning the Kitchen Counter with a glove

By: James Peacock

Listeria monocytogenes bacteria are an especially dangerous form of foodborne pathogen. They have the ability to survive and even thrive, in very cold environments. This means that freezing the bacteria will not kill them, nor will it hamper their growth. Listeria monocytogenes first became known as a foodborne illness in 1981. Although it had received previous study, it was not until a major outbreak in Canada that the pathogen began to gain more attention. The CDC began to track Listeria infection more than 30 years ago. The CDC now estimates that there are about 1600 cases of illness caused each year by Listeria bacteria. This leads to about 260 deaths per year. Several risk factors make an individual more likely to develop a Listeria infection. Newborns, older adults, and people with suppressed immune systems are all at an increased risk. Pregnant women are also about 10 times more likely to develop a Listeria infection.

The testing process for a Listeria infection is a simple tissue sample used to foster the growth of a bacterial culture. If Listeria is seen in the culture, then the individual who provided the sample has tested positive for a Listeria infection. This infection is usually treated with antibiotics. The Listeria poisoning itself may not produce symptoms for up to two months after the infection, although symptoms usually present within 3-10 days. Listeria infections often produce similar symptoms to other foodborne illnesses, including fever and diarrhea. However, if the infection becomes invasive, meaning it has left the gut, the symptoms can worsen. In pregnant women with invasive Listeria, fever, fatigue, muscle aches, and diarrhea are all common symptoms. However, Listeria has been known to cause miscarriage, stillbirth, infection in newborns, or premature delivery. In individuals with invasive Listeria who are not pregnant, fever, diarrhea, headache, stiff neck, confusion, convulsions, loss of balance, and muscle aches are all common symptoms.

Because a Listeria infection can be deadly, healthcare professionals often rely on a prompt diagnosis in order to help treat the infection before it becomes serious. Listeria identification tests are also important in the food production industry, as quick testing can help prevent contaminations, recalls, and Listeria outbreaks. Researchers also work to find new and more effective ways to test for Listeria. Though cultures remain one of the most common ways to diagnose and identify Listeriosis, they can take up to about five days to grow the proper number of bacterial colonies required to positively diagnose Listeriosis. In the 1990s, a process called a polymerase chain reaction (PCR) was developed. This process uses heat to break the weak hydrogen bonds that hold strands of DNA together. Polymerase, the enzyme that synthesizes DNA by adding the matching base pairs to a pre-existing half strand of DNA, then completes the separated strands of DNA to make two separate strands, effectively doubling the amount of DNA. Extra base pairs are provided for the polymerase in a solution added during the PCR test. The process of splitting and rebuilding the DNA repeats for about 40 hours, and results in thousands of copies of the same segment of DNA. This process can be prone to errors, though, and is not as versatile as cultures in the types of samples that can be run through it. In the recent weeks, two different companies have announced new ways to test for Listeria bacteria, both of which promise to increase the accuracy or speed of bacterial testing. Both of these methods identify Listeria bacteria using rRNA rather than DNA.

Ribosomal Ribonucleic Acid

Ribonucleic Acid (RNA) is very similar to DNA. In fact, out of the four different nucleotides used in DNA—Adenine, Thymine, Guanine, and Cytosine—there is only one that is not also in RNA. RNA uses the nucleotide Uracil instead of Thymine. The three types of RNA are mostly used in the creation of proteins. Over the course of the natural function of the cell, proteins must be created. Proteins are absolutely vital to the human body, participating in everything from immune response to literally holding the body together. The recipe for these proteins is found in the DNA of the cell. When a cell produces a protein, DNA is changed into messenger RNA (mRNA) through the process of transcription. Transcription works in a similar way to the process involving polymerase described above. Instead of DNA polymerase matching base pairs and binding two strands of DNA together, though, RNA polymerase matches base pairs to produce a separate, single strand of mRNA. The mRNA is then moved to the cytoplasm, where it undergoes the process of translation. In this second step of the protein-making process, the mRNA binds to ribosomes found in the cell. The ribosomes facilitate the creation of a protein by pairing the mRNA with the corresponding transfer RNA (tRNA). A molecule of tRNA has three base pairs in it called an anticodon and has a specific amino acid attached to the top of the molecule. When a tRNA’s anticodon is paired with the correct three base pair set in the mRNA—a codon—the amino acid attached to the tRNA is added to the protein. When the right numbers of amino acids are bonded together, a protein has been created. There is a third type of RNA, called ribosomal RNA (rRNA), which is found inside the ribosome. The rRNA is created in the nucleolus, the denser core of the nucleus. It’s the rRNA that is important in the recently developed tests. Because the rRNA is made from DNA in the nucleus, it will be able to identify the pathogen it originated from in the same way that DNA would. RNA is also much more plentiful in the cell, meaning that while there may only be one copy of a specific strand of DNA, there may be thousands of copies of rRNA that can be used to identify a pathogen.

Roka Bioscience

Roka Bioscience recently announced that they had received AOAC Certification for their Atlas system. The Atlas system uses rRNA detection to find Listeria in a sample taken from the environment. The system represents advancement in not only the speed at which Listeria can be detected but also the accuracy of testing. Again, traditional culture methods of testing take about 5 days to produce a sample. PCR testing takes about 2 days to replicate enough DNA to allow for identification. The Atlas system boasts a test result speed of about a day, making it one of the fastest tests around. In a test done by Roka, samples were taken from various types of eggs, including frozen, dried, and salted egg whites. The samples were put into cultures, PCR, and in the Atlas System. With the culture samples acting as a confirmation, the PCR test was able to identify Listeria with a false negative rate of almost 28%. The Atlas system did not record a single false negative. The Atlas System recently received AOAC certification for producing an accurate Listeria test after between 18 and 24 hours.

Neogen

Neogen has also made waves recently because of a new test that they have developed for detecting Listeria. The test works in a similar way to the Atlas system, in that it focuses on rRNA rather than DNA. The test works by placing a swab with a sample on it into a test tube. The test tube contains a lysis buffer compound, which tears up bacteria, releasing what is inside. Reagents in the tube, if Listeria rRNA is identified, multiply the rRNA to make it detectable. What sets the Neogen test apart from other methods is that there is no sample enrichment necessary for the process to detect Listeria. The test can process a sample in about an hour. This is a much faster test than most methods. Early studies show that the Neogen test is just as sensitive as enrichment-based cultural methods. The process is currently being tested by the AOAC in an effort to confirm the accuracy of Listeria detection.

Conclusion

Both of these tests offer the chance for health officials and companies to more accurately and more quickly detect the presence of Listeria monocytogenes bacteria. If the tests are more accurate and faster, it will be much easier to prevent potential contaminations. Either through the increased effectiveness of recalls or through the prevention of those recalls in the first place, companies can prevent foodborne illnesses. Increasing effectiveness of testing can also help investigators pinpoint the source of a foodborne illness outbreak much quicker, making it more likely that they can stop an outbreak from spreading. Advances in rRNA detection may even be useful in identifying pathogens other than Listeria.

Sources:

http://www.feedstuffs.com/new-products/neogen-develops-rapid-test-listeria

http://www.prnewswire.com/news-releases/roka-bioscience-announces-aoac-certification-for-18-hour-sample-enrichment-using-acterotm-elite-listeria-enrichment-media-and-the-atlas-listeria-environmental-assay-300485949.html

https://www.streetinsider.com/Corporate+News/Roka+Bioscience+%28ROKA%29+Receives+AOAC+Certification+for+Sample+Enrichment+using+Actero+ELITE+Listeria+Enrichment+Media+and+Atlas+Listeria+Environmental+Assay/13087409.html

https://www.zacks.com/stock/news/267166/neogen-introduces-test-for-listeria-in-environmental-samples

https://www.nobelprize.org/educational/medicine/dna/a/translation/rrna_synthesis.html

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