Development & Evaluation of PCR and Elisa kit


Enzyme-linked immunosorbent assay-polymerase chain reaction (PCR-ELISA) is an immunodetection method that can quantify the PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to the conventional PCR method, with a shorter analytical time and lower detection limit.

Its high specificity and sensitivity, coupled with its semi-quantitative capability, give it enormous potential to serve as a powerful screening tool in various industries, such as medical, veterinary, and agricultural. With recent advances in kits-de-PCR-e-ELISA, the assay is expected to become more widely recognized for its rapid and sensitive detection limit, which could improve overall diagnostic time and quality.

Comparisons of PCR-ELISA with other PCR-based molecular approaches

Since the introduction of this tool, several studies have been conducted to compare the performance of PCR-ELISA with other tools. Many agreed that detection of DIG-labeled products by microwell capture hybridization assay makes PCR-ELISA a more sensitive tool than agarose gel electrophoresis analysis because specific hybridization and enzymatic staining increase. the positive signal from biotin-labelled, probe-bound PCR products.

PCR amplicons are analyzed using a colourimetric assay; thus, not only is there a reduced risk in the use of mutagenic staining materials and a significant reduction in possible DNA contamination, but it also allows the method to serve as a semi-quantitative tool. As this detection uses gene-specific probes for detection, the specificity of the tool is highly emphasized. Not only can samples with that particular gene be detected, but they can also be quantified based on colour intensity.

The presence of a higher colour intensity indicates that more of the probe is bound to the specific gene sequence, forming a hybrid complex that then bound peroxidase-conjugated anti-DIG antibody and the colourimetric peroxidase substrate ABTS, allowing the detection. While PCR-ELISA cannot provide an accurate estimate of the actual gene of interest that is present compared to real-time PCR (qPCR), it does provide a quick summary of whether a particular substrate is high or low at a particular time point as colourimetric detection. is directly proportional to the number of the predicted gene of interest.

Another main attraction of PCR-ELISA is that the assay allows for large-scale screening using only standard laboratory equipment, making it suitable for use in clinical laboratories. This should serve as another incentive for labs with fewer resources, as a survey by Comley showed that respondents do not resort to fully automated ELISA equipment despite its availability, possibly due to high purchase and cost costs. equipment maintenance.

Last but not least, the overall analytical time of this assay is also much shorter than the conventional PCR method, making it a promising tool for future uses, especially when dealing with large sample sizes. With new discoveries and new inventions, molecular biology tools need to be continually improved and developed for faster and more efficient results.

Every new technology that was developed has its pros and cons compared to other technologies. If the study involves an unknown gene, then conventional PCR with agarose gel detection would be the only option available, since both qPCR and PCR-ELISA require the development of primers and/or probes that are difficult to achieve. in a new target gene.

PCR-ELISA applications

With the aforementioned advantages of PCR-ELISA and its semi-quantitative capability, several researchers propose the use of PCR-ELISA in a wide range of fields, from basic detection and diagnosis to quality control and quantitative monitoring of infectious diseases, detection of food allergens, phytopathogens and biomarkers, detection being its main application.

  • Detection and Diagnosis

Due to its high sensitivity and specificity, several studies on the use of PCR-ELISA as a detection and diagnostic method have proven to be successful. Since rapid diagnosis in the medical field can affect the life or death of the public, the articles below are among some of the recent studies reported in the last 5 years on the detection of various diseases and pathogens in medical diagnosis: identification of cancer cells; screening for the presence of hepatitis types A, B, C, and E; species detection and species identification of dermatophytes; invasive fungal infections in immunocompromised patients; detection of poliovirus, enterovirus, and norovirus; and determination of blood group antigens for hemolytic disease in cases of newborns and polytransfused patients.

There are also a number of publications using PCR-ELISA in the food industry, such as the detection of harmful foodborne pathogens such as Campylobacter sp., Salmonella, Listeria monocytogenes, Escherichia coli, Brucella melitensis and Vibrio parahaemolyticus. Without limiting the use of the method in the medical and food industry, the study of PCR-ELISA extends even to the veterinary industry.

Studies include the detection of the Leishmania parasite and the detection of various avian viruses in chickens. Other screening studies that help detect the presence of plant pathogens include tomato spotted wilt, potato spindle tuber, the prevalence of each phylogenetic group among infected grapevine varieties and plant viruses in woody species. plants. PCR-ELISA can also detect the presence of harmful waterborne pathogens in both supply water and industrial cooling tower water.

  • Quantitative Monitoring

Many studies also suggest the use of the assay for quantitative control as a quick indication of the presence or absence of a particular substrate and its estimated concentration. It is a very important tool, especially for immunocompromised patients who are sensitive and susceptible to their environment, as it allows determining the appropriate level of antiviral management.

Among the studies on the monitoring of quantification by PCR-ELISA are the evaluation and monitoring of cytomegalovirus infection in bone marrow transplant recipients, the diagnostic value of the combined determination of telomerase activity in sputum induced, pleural effusion and fiberoptic bronchoscopic biopsy in patients with lung cancer, and quantitative monitoring of the Leishmania parasite in cattle.