RT-PCR Test for COVID 19

Less than a decade after the last human outbreak caused by a zoonotic coronavirus, the Middle East respiratory syndrome (MERS) in 2012, a novel coronavirus spillover had emerged in China in 2019 and now we are witnessing a deadly pandemic. A year later, India is now facing the second wave of covid 19, the nation is struggling hard to breathe out. Widespread testing for the SARS-CoV-2 virus is important to both slow the velocity of the virus and gain information about how widespread it is.

According to the regulatory authorities such as the FDA, Food & Agriculture Organization of the United Nations (FAO), RT-PCR is one of the most accurate laboratory testing methods for the current coronavirus pandemic. 

The real-time Reverse transcription-polymerase Chain Reaction (RT-PCR) test is primarily based on PCR (Polymerase Chain Reaction), a process that repeatedly copies and amplifies the specific genetic fragments of the virus, ensuring that there is enough of a sample to conduct the analysis.

Polymerase Chain Reaction (PCR) Working Principle

The principles behind every PCR are the same. Five cores ‘ingredients’ are required to set up a PCR, which are –

  1. The DNA template to be copied.
  2. Primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the section of DNA you want to copy.
  3. DNA nucleotide bases(also known as dNTPs). DNA bases (A, C, G, and T) are the building blocks of DNA and are needed to construct the new strand of DNA.
  4. Taq polymerase enzyme to add in the new DNA bases.
  5. Buffer to ensure the right conditions for the reaction.

How does the RT-PCR test work?

PCR involves a process of heating and cooling called thermal cycling which is carried out by a PCR machine.

  • There are three main stages:
  1. Denaturing– when the double-stranded template DNA is heated to separate it into two single strands.
  2. Extending – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
  3. Annealing ­– when the temperature is raised, and the new strand of DNA is made by the Taq polymerase enzyme

These three stages are repeated 20-40 times, doubling the number of DNA copies each time. So by the end of the reaction, the size of the new strand becomes 220-240 of the initial strand. Reverse transcription PCR, or RT-PCR, allows the use of RNA as a template. An additional step allows the detection and amplification of RNA. The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The quality and purity of the RNA template are essential for the success of RT-PCR. The first step of RT-PCR is the synthesis of a DNA/RNA hybrid. The single-stranded DNA molecule is then completed by the DNA-dependent DNA polymerase activity of the reverse transcriptase into cDNA. The efficiency of the first-strand reaction can affect the amplification process. From here on, the standard PCR procedure is used to amplify the cDNA. The possibility to revert RNA into cDNA by RT-PCR has many advantages. RNA is single-stranded and very unstable, which makes it difficult to work with.

The PCR machine in which the Polymerase Chain Reaction takes place is known as a Thermal cycler. It works with all the necessary chemicals. During the process, one of the processing chemicals produces a fluorescent signal which only gets activated above the threshold limit of amplification, i.e., only if the coronavirus is present in enough quantity in the body.

Steps Involved in RT-PCR Test

There are essentially three steps for an RT PCR test:

Step-1 Sampling

The RT-PCR test starts with a simple swab taken from inside a person’s throat or nose. A swab contains a soft tip on a long, flexible stick that is inserted into the nose of the person, who is to be tested. There are different types of nose swabs including nasal swabs that collect a sample immediately inside your nostrils and nasopharyngeal swabs that go further into the nasal cavity for collection. Either type of swab is sufficient for collecting material for the COVID-19 PCR test. After collection, the swab is sealed in a tube and then sent to a laboratory.

Step-2 Extraction

When a laboratory technologist receives the sample, they perform a process called extraction, which isolates genetic material from the sample including genetic material from any virus that may be present. Coronaviruses have RNA or ribonucleic acid as their genetic material. However, swabs from patients yield only a tiny quantity of RNA, which is not adequate for the testing process.

To overcome this problem, the RNA — a single-strand molecule — is converted into double-stranded DNA using an enzyme. This is known as reverse transcription.

Step-3 Polymerase chain reaction

Researchers select specific areas in the genome that do not mutate rapidly as the virus evolves and create copies of these using the PCR process.

 

Interpreting RT- PCR test results

positive test result means that it is very likely that the person has COVID-19. Most people have mild illnesses and can recover safely at home without medical care. It is advised to contact the healthcare providers if the symptoms get worse or if any questions or concerns arise.

negative test result means the person probably did not have COVID-19 at the time he/she took the test. However, it is possible to be infected with SARS-CoV-2 but not have enough virus in the body to be detected by the test. For example, this may happen if a person recently became infected, but does not have symptoms, yet; or it could happen if one has had COVID-19 for more than a week before being tested. 

Ups and Downs of COVID-19 PCR test

The main advantages of the COVID-19 PCR test are its accuracy and reliability. It is the most accurate test available for COVID-19 detection.

But it also has its downsides. Because the test can detect exceedingly small amounts of virus material, it can continue to detect fragments of the SARS-CoV-2 virus even after you have recovered from COVID-19 and are no longer contagious. So, people may continue to test positive even if they have had COVID-19 in the distant past, even though they cannot spread the SARS-CoV-2 virus to others.

Comparison with Antigen Test

Diagnostic tests:

  1. PCR test: This tests for the presence of the actual virus’s genetic material or its fragments as it breaks down. This is the most reliable and accurate test for detecting active infection.
  2. Antigen test: This test detects bits of proteins on the surface of the virus called antigens. Antigen tests are typically considered rapid, taking only 15 to 30 minutes but are less accurate than a PCR test. Rapid antigen tests are most accurate when used within a few days of the start of your symptoms, which is when the largest amount of virus is present in your body. Because this test is not as accurate as a PCR test, if an antigen test is negative, the healthcare provider may order a PCR test to confirm the negative test result.

The whole world is still struggling hard in the pandemic. The RT-PCR test plays a keys role to monitor the current situation. The accuracy of test results is really important in the first-line defense.  RT-PCR is currently the most accurate test which is used in the covid 19 diagnosis tests, but like any other tests, it is also not 100% accurate. The minimum sensitivity (ability to detect positives) demanded by the Indian Council of Medical Research (ICMR) for validating an RT-PCR test is 95%. That means up to 5% false-negative results are expected. Experts recommend Covid-19 treatment for everyone showing classic symptoms irrespective of RT-PCR results.

Reference

  1. https://www.apollohospitals.com/covid-19-rt-pcr-test/
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC154784/
  3. https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction
  4. https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing
  5. https://theconversation.com/how-does-the-coronavirus-test-work-5-questions-answered-133118
  6. https://www.thehansindia.com/hans/opinion/news-analysis/covid-19-human-perspective-response-685138
  7. https://theprint.in/science/rt-pcr-antigen-antibody-truenat-all-you-need-to-know-about-the-different-covid-tests/448733/
  8. https://theprint.in/opinion/antibody-test-or-rt-pcr-both-needed-to-fight-covid-19-dont-rake-up-controversies/390290/

 

Carbon Dioxide Processing and Milk

Thermal treatment of food product is always related to high impact on their nutritional and organoleptic values and a times are not desirable specially in case of thermally sensitive food products. Most general practice in dairy industry is having high temperature short time (HTST) pasteurization practice where the liquid milk is subjected to high temperature having range of 70-78 Deg. C, which although reduce microbial load in milk but the milk is in risk of losing thermally sensitive nutrients and enzyme, off flavor development due to overheating and other changes in organoleptic factors.

High pressure carbon dioxide, also known as HPCD is an emerging technology being used for thermally sensitive food product. A type of pasteurization that can be an alternative to thermal pasteurization in food industry. This technique is considered as an alternate to pasteurization as this process has been known for bactericidal effect.

Solubility of Carbon Dioxide-

The amount of carbon dioxide in any solution is known as the concentration of carbon dioxide. There is always saturation level, i.e., the maximum amount of carbon dioxide that can be dissolved in any liquid, present which is exploited during processing. Carbonation is the term given to the process of addition of carbon dioxide in liquid.

During the milking stage, milk already consists of certain percentage of CO2 ~5 millimole but with time, the concentration drops as it equilibrate with the atmospheric CO2. The solubility of CO2 in milk is controlled by temperature and product viscosity. The more the temperature, less the diffusion of CO2 in milk but increase in viscosity helps in diffusing more gas in higher temperature product. With slight infusion of CO2, freezing point of milk can be brought down but is reversible until the infusion is moderate.

How Microbes react to HPCD-

What makes the CO2 to be antimicrobial? Well, the acidification property and solvent properties helps in attaining the goal, and maximum effect can be attained in the supercritical state. The liquid form of CO2 has greater solubility and can penetrate through the microbes and disrupts the cells. The acidifying property when is doubled with pressure the bactericidal effect. Depending on applying temperature and pressure, impact on microorganism can be altered

  1. At low pressure and low temperature carbon dioxide can inhibit the growth of pathogenic microbes and alters the pH of the medium which infers double attach to the growth of microbes.
  2. At high pressure and moderate temperature, carbon dioxide tends to attack the microorganism and inactivates by destroying its cells.

Parameter influencing –

The process parameters such as temperature, pressure, time of exposure and agitation affects the overall shelf life of product in case of carbon dioxide processing.

  1. Temperature- Higher temperature facilitates the higher diffusion and penetration of carbon dioxide in the microorganism cells leading to more lethal effect on present microorganisms. But the optimal range is to be kept in mind while providing heat to the product as the higher temperature leads to less solubility of carbon dioxide in product. From past research it has been evident that temperature range of about 20- 45 Deg. C is optimal for carbon dioxide treatment.
  2. Pressure- When pressure is increased it has been noted that less exposure time is required for the deactivation of microorganism. Also fast depressurization also helps in the cause.
  3. Agitation- In case of no agitation, only the periphery microorganism shall come in contact with the carbon dioxide present in product hence, constant agitation facilitates the contact of carbon dioxide and microorganism hence high chances that the all microorganism in product is deactivated.

There has been almost hundreds of bacteria identified in the raw milk. All have different characteristics, and each have differently impacted when subjected to various processing conditions. The product composition also have important role to play on the shelf life and treatment effects. The statement is backup with various previous and ongoing studies. Addition of certain amount of starch didn’t affected the bactericidal effect of CO2 while presence of whey protein affects negatively on the efficiency of carbon dioxide processing. Also lower water content also reduces efficiency of CO2 treatment as it act as a protective barrier for microbial cells. Salt also affects the kill efficiency- Lower salt reduces the potency while higher salt concentration facilitates the kill efficiency of carbon dioxide processing.

To conclude, listing some of the attributes that can benefit the commercializing of carbon dioxide processing of food products.

  1. Low fat content
  2. High Water activity
  3. Low to moderate sugar content
  4. Alcohol and protein content
  5. Low pH and
  6. Low viscosity

Skimmed liquid milk fits precisely in this category and thick dairy products are hard to treat with the carbo dioxide pasteurization technique.

All studies till date conducted, including product shelf life, effect on food product, and others are of laboratory level which is the holding back point for this technology. Need of the time is to carry out extensive research on the utilization in commercial level so that the benefits can be fully commercialized.

Pulse-Electric-Field-PEF-Food

Pulsed Electric Field

Food-processing industry has made large investments in processing facilities relying mostly on conventional thermal processing technologies with well-established reliability and efficacy. Food contains many heat sensitive nutrients which include vitamins, minerals, and nutrients having functional properties such as pigments, antioxidants, bioactive compounds. Many processes during manufacturing of food cause detrimental effects on these nutrients. Therefore, retention of these nutrients in food products requires innovative approaches for process design because of their sensitivity to a variety of physical and chemical factors, which causes either loss of biological functionality, chemical degradation and premature or incomplete release. Alternative methods for thermal processing of food are gaining importance, due to increased consumer demand for new methods of food processing that have a reduced impact on nutritional content and overall food quality. Also because of the increasing consumer demand for minimally processed fresh-like food products with high sensory and nutritional qualities, there is a growing interest in non-thermal processes for food processing and preservation.

Pulsed Electric Field (PEF) is one such widely used novel non-thermal technology for food preservation. PEF is a mild food preservation technique making use of short electric pulses of variable intensities yielding few to no detrimental effects on quality attributes in pumpable foods such as fruit juices, liquid eggs, jams, soups, milk, yogurt etc., but with advancement, this technology have become friendly for solid foodstuff too.

Working Principle

The process is based on electroporation, which generates high voltage (10–80 kV/cm) pulses into foods placed between two electrodes for short time at ambient temperature, then packaged aseptically and distributed in refrigerated condition. Food is exposed to electrical field of treatment chamber leading to the opening of pores in tissue mass. This process attains a 5 log reduction on most pathogenic bacteria by rupturing the cell membranes in liquid media. Inactivation of microorganisms occurs due to generation of shock wave generated by an electric arc that stimulate the formation of highly reactive free radicals from chemical species in food. It causes only minimal detrimental changes to the physical and sensory properties in foods, helping in retaining ‘fresh’ quality and assists in nutrient retention.

Treatment Affecting Factors  

  • Electric field strength- Strength of electric field depends on type of food and microorganism to be treated.
  • Processing time- Varies with microorganism.
  • Treatment temperature
  • pH-Low acidic foods are preferred but not limited to them
  • Ionic Strength
  • Conductivity of the medium
  • Type of microorganism present in food media

Construction:

Construction of PEFs machine involves a pulsed power supply, treatment chamber that converts the pulsed voltage into PEF and a control panel

  • High voltage Pulse Generator: This unit provides a maximum of 2.5 kV pulses. The instrument consists of a capacitor (7 F), charge and discharge switches, and a wave controller. The wave controller may be connected to the electroporator to improve the discharge pattern. Treatment cuvettes with a 0.1-cm electrode gap and l00 L volume may be used for PEF treatments, which give a maximum intensity of approximately 25 kV/cm. Appropriate voltage and current monitors should be attached to it.The frequency, shape and amplitude of the pulses result in the intensity of the treatment.
  • Treatment chamber: PEF treatment chamber may be static or continuous which consists of two electrodes, with insulating materials in between that acts as an enclosure containing food materials. Uniform electric fields can be achieved by parallel plate electrodes with a gap sufficiently smaller than the electrode surface dimension. The chamber also contains a spacer, and two lids. Each electrode is made of stainless steel, whereas the spacer and lids are made of polysulfone. The distance between two electrodes is termed as the treatment gap of the PEF Chamber. A flow channel was provided between the two electrodes to eliminate dead corners as well as to ensure uniform treatment. The electric field may be applied in the form of exponentially decaying square waves, bipolar, oscillating pulses at ambient, sub ambient, or slightly above ambient temperature. Cooling of the chamber is accomplished by circulating water at a selected temperature through jackets built into the two stainless steel electrodes.
  • Control System for monitoring Process Parameter: It consists of two major devices:
    1. Oscilloscope: It measures the voltage across the treatment chamber and shows the output voltage shape.
    2. Temperature Probe: Sample temperature is measured with a thermal probe placed inside the treatment chamber.

Field of Applications

  1. Improved quality of Potato chips and finger fries with less fat content: PEF pre-treated potatoes enables cutting at optimum product texture which results in smooth surfaces, reduced starch loss, less damage and less absorption of oil/fat by potato chips and fries.
  2. Drying: PEF facilitates rapid removal of moisture from cell matrix.
  3. Wastewater Treatment: PEF burst cells are more susceptible to other biological and chemical processes such as anaerobic digestion, which significantly reduces solid waste content in wastewater. Thus reducing the treatment time for wastewater.
  4. Efficient Extraction of Fruit Juices: Reports suggests, PEF treated apples and carrots are found to be producing more juice as their cell mass gets disintegrated which is the essential pre requisite for solid liquid separation during juice extraction from fruits and vegetables.

As this process include wide benefits like maintaining the product quality without thermal and mechanical stress, not degrading the nutrient properties, not producing any harmful by product, increasing the extraction yield by softening of tissues, it involves certain limitation such as its limited effects on microbial spores and its ineffectiveness on foods that have higher or variable electrical conductivity. Traditional thermal processing techniques for preserving foods leads to destruction of heat sensitive ingredients such as vitamins, which is helpful in promoting health. Therefore, PEF can be seen as the promising replacement, which allows the preservation of heat sensitive food products for which fresh taste is the main quality parameter, without or with only minor detrimental effects on the quality.

Foreign_Material_Control_by_Metal_Detector

Foreign Material Control

Foreign material render to food adulteration, and hence is concern of food safety and food quality. Foreign material is any extraneous matter, whether of a physical, chemical or biological nature, found in food. Usually foreign bodies make the food unfit for human consumption. It is important to understand what can be foreign material in their processing line. Documenting all the possible foreign material, their root cause, source of entry, their control or preventive measure. Validation and verification of such data become easy and can be good communicable platform that can be used between management and employees.

Need to Control Foreign Material

Foreign matter renders to food adulteration and any incidence of foreign matter harms the consumer, undermines confidence in the brand, arising questions on the quality of food product being manufactured. It is one of the important factor in effective implementation of the company’s food safety program, particularly the Hazard Analysis Critical Control (HACCP) system. Classified on the basis of the level of control a food processor can exercise to eliminate the risk the foreign materials in food.

  • Low Risk: If good control measures have been established, but minor infractions occur.
  • Medium Risk: If some control measures were established, but inconsistencies occur.
  • High Risk: If little or no control was established, major and critical infractions occur.

Sources of foreign Material

Sources of foreign material in the food facility has been concised to five basic sources

  1. Inadvertent from the field (stones, metal, insects, undesirable vegetable matter such as thorns or wood, dirt, or small animals).
  1. Inadvertent resulting from processing and handling (bone, glass, metal, wood, nuts, bolts, screening, cloth, grease, paint chips, rust, and so on).
  2. Materials entering the food during distribution, such as insects, metal, dirt, or stones.
  3. Materials intentionally placed in food (employee sabotage, adulteration).
  4. Miscellaneous materials, such as struvite and other materials in this class

Type of Hazard & its

Type of hazard that are common in food segment are, but not limited to,

  • Glass: Sharp glass contamination often occurs during filling processes in glass containers if a container is accidentally broken. Another source, but less frequent, is light bulbs broken during building maintenance.
  • Metal: Sharp metal objects may include screws and equipment splinters, blades, broken veterinary needles, fragments and clippings of prior processing procedures.
  • Plastics: Soft and hard plastics may come from packaging material of an intermediary production phase.
  • Wood: Wood splinters may have their origin at the farm or may come from handling wooden pallets.
  • Stones: Small stones are common in crops like peas or beans contaminated during harvest.

Control: Controls for such hazard include inspection of raw materials and ingredients, looking for field contaminants, provision of good storage facilities, usage of lamp covers and light protector to avoid bulb breakage and maintenance of effective pest control, development of specifications and controls for all ingredients and components, effective detection and elimination systems for physical hazards, such as metal detectors or magnets to remove metal particles; use of X-ray and low-power microwave systems to detect nonmetallic materials, periodic training of employees engaged with shipping, receiving, storing, handling and equipment maintenance that encompasses the entire food production chain. Pallets coming in contact with processed food of food grade material, exempting employees from taking any accessories such as rings, bands, hairclips, watch, lens, jewelry etc. in the processing section, and to have proper SOPs that need to be maintained and should be trained. Other things that can be included are proper cleaning and maintaining sanitization in and around processing areas.

Detection Technology

  1. Metal detectors: Metal detectors find splinters from machinery, fractions of broken cutters and blades, needles, screws or fragments of clips, jewelry, accessories, and other foreign materials.
  1. X-ray detectors: These respond to metal, stone, bone, hard plastics and Teflon. Both systems can screen the product after the filling procedure. X-ray detectors may find glass pieces that result when a jar or bottle is crushed during malfunction of the packaging line.
  2. Near-field radar response: Foreign bodies are detected in embedding material by transmitting low-power microwaves through the material. The system uses a microwave sensor designed for emulsions and pumpable products. It measures the dielectric properties of the food flow of processing equipment. Foreign materials produce a deviation from the norm, activating a pneumatic rejection unit.

From initial stages only like receiving operations of raw agricultural commodities, inspection and wide range of cleaning steps are involved to remove undesirable materials such as leaves, stones, twigs, mud and different kinds of creatures. Hence it is recommended that processors should design process line and equipment in accordance to the need and type of hazard that can infiltrate food product.