Salmonella (SS) real – time fluorescence quantitative PCR detection kit

I. Definition

The Salmonella (SS) real – time fluorescence quantitative PCR detection kit is a tool used to detect the presence of Salmonella in samples and perform quantitative analysis on it.

II. Detection Principle

1. Principle of real – time fluorescence quantitative PCR
   • Based on the polymerase chain reaction (PCR) technology, primers and fluorescence probes designed for the specific gene sequences of Salmonella are added to the reaction system.
   • When Salmonella is present in the sample, during the PCR reaction process, the primers will specifically bind to the DNA template of Salmonella. Under the action of DNA polymerase, chain elongation occurs using dNTP (deoxyribonucleoside triphosphate) as raw materials.
   • The fluorescence probe does not emit fluorescence in its intact state. As the PCR reaction proceeds, Taq enzyme (a type of DNA polymerase) will cut the fluorescence probe during the elongation process, releasing fluorescence signals.
   • With each PCR cycle, there is a corresponding increase in the fluorescence signal. By detecting the intensity change of the fluorescence signal, the PCR reaction process is monitored in real – time, thereby determining the content of Salmonella in the sample.

III. Kit Components

1. Reaction buffer
   • It provides a stable chemical environment required for the PCR reaction, including maintaining an appropriate pH value and ion concentration, etc., ensuring that enzymes such as DNA polymerase can function normally.
2. Primers and fluorescence probes
   • The primers are short – chain DNAs designed according to the specific gene sequences of Salmonella and are used to guide DNA synthesis. The fluorescence probe is an oligonucleotide chain with a fluorescent reporter group and a quenching group and is used to detect the amplification of Salmonella DNA during the PCR reaction.
3. DNA polymerase
   • Such as Taq enzyme, it is a key enzyme for catalyzing the elongation of DNA strands. It can add dNTPs one by one to the newly synthesized DNA strand according to the sequence of the template strand on the basis of primers.
4. dNTP
   • It is the raw material for synthesizing new DNA strands, including dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (deoxyuridine triphosphate), providing the material basis for DNA strand synthesis in the PCR reaction.
5. Positive and negative controls
   • The positive control is a sample containing a known amount of Salmonella DNA and is used to verify the effectiveness of the kit and the accuracy of the detection process. The negative control is a sample without Salmonella DNA and is used to exclude false – positive results, ensuring the reliability of the test results.

IV. Types of Test Samples

1. Food samples
   • Including meat, eggs, milk, and their products, etc. For example, in the meat – processing process, Salmonella contamination may occur. By collecting meat samples for testing, it can be determined whether the product meets food safety standards.
2. Environmental samples
   • Such as soil, water, countertops, and other environmental samples from breeding farms, food – processing workshops, kitchens, etc. Salmonella may exist in these places, and testing environmental samples helps to understand the pollution status of the environment and provides a basis for hygiene management and prevention and control measures.
3. Clinical samples
   • Mainly feces, blood, urine, and other samples from patients. For patients suspected of being infected with Salmonella, testing their clinical samples can assist in diagnosing diseases so that treatment measures can be taken in a timely manner.

V. Detection Steps

1. Sample processing
   • Food samples need to undergo operations such as crushing and homogenization to fully release the possible Salmonella. Environmental samples may need pre – treatment such as enrichment culture to improve the detection sensitivity. Clinical samples need to be processed according to corresponding medical testing specifications. For example, fecal samples may need dilution operations.
2. DNA extraction
   • Extract the DNA of Salmonella from the processed samples. Usually, chemical reagent methods or kit methods are used to release the DNA of Salmonella from cells and remove impurities to obtain a relatively pure DNA solution.
3. Preparation of the reaction system
   • According to the kit instructions, mix the reaction buffer, primers, fluorescence probes, DNA polymerase, dNTP, and extracted sample DNA (or control samples) in a certain proportion to form a PCR reaction system.
4. PCR reaction
   • Place the prepared reaction system into a real – time fluorescence quantitative PCR instrument, set appropriate reaction programs, including temperature and time parameters for steps such as pre – denaturation, denaturation, annealing, and elongation, and start the PCR reaction.
5. Result analysis
   • Analyze the curve of the change in fluorescence signal intensity with the number of cycles through the software of the real – time fluorescence quantitative PCR instrument, and determine the content of Salmonella in the sample according to the standard curve (generated by positive control samples).

VI. Application Fields

1. Food safety testing
   • In the production, processing, and circulation of food, quickly and accurately detect Salmonella in food to ensure consumers’ food safety. For example, conduct random inspections of fresh food in supermarkets to ensure that the food on the shelves meets hygiene standards.
2. Environmental hygiene monitoring
   • Used to monitor Salmonella contamination in various environments and guide environmental hygiene cleaning and disinfection work. For example, regularly test the kitchen environment of school cafeterias to prevent food – borne disease outbreaks due to environmental problems.
3. Clinical diagnosis
   • Assist doctors in diagnosing diseases caused by Salmonella infections and provide a basis for formulating reasonable treatment plans. For example, for patients with unexplained diarrhea, detect Salmonella in feces to determine the cause.