Monkeypox Information

2. What is Reverse Transcription Polymerase Chain Reaction (RT-PCR)?

Reverse transcription-polymerase chain reaction is a molecular technique commonly called RT-PCR. PCR stands for polymerase chain reaction, a laboratory method used to amplify specific regions of DNA for diagnosis and analysis in medical research. Reverse transcriptase PCR, instead of DNA, uses mRNA as the starting template.

Real-time RT-PCR is a modified version of PCR in which the enzyme called reverse transcriptase is used. This enzyme facilitates the conversion of an RNA sequence into its complementary DNA sequence. RT-PCR works on this principle of reverse transcription. It is a real-time technique that combines reverse transcription of RNA into DNA and the amplification of target DNA. It’s a qualitative means of detecting a particular RNA sequence with a high degree of accuracy and sensitivity.

The Monkeypox virus nucleic acid Test Kit is performed on the applicable instrument to detect DNA extracted from lesion exudate specimens. It utilizes the monkeypox virus conserved sequence of F3L gene as multiplex real time PCR amplification target regions.

The fluorescent quantitative PCR instrument can automatically draw a real-time amplification curve based on the detected fluorescent signal, so as to realize the qualitative detection of the monkeypox virus at the nucleic acid level. The multiplex real time PCR detection system includes a pair of primers and probes for internal control (RNase P). The positive control and negative control included in the kit can be used as an external control in every test run. The results of internal and external control can be used to monitor correct specimen collection, handling and real time PCR process.

3. What is Reverse enzyme linked immunosorbent assay (ELISA)?

ELISA is an antigen antibody reaction. It is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. An enzyme conjugated with an antibody reacts with colorless substrate to generate a colored product.

ELISAs are typically performed in 96-well polystyrene plates. The serum is incubated in a well, and each well contains a different serum. A positive control and a negative control would be included among the 96 samples being tested. Antibodies or antigens present in serum are captured by corresponding antigen or antibody coated on to the solid surface. After some time, the plate is washed to remove serum and unbound antibodies or antigens with a series of wash buffer. To detect the bound antibodies or antigens, a secondary antibodies that are attached to an enzyme such as peroxidase or alkaline phosphatase are added to each well. After an incubation period, the unbound secondary antibodies are washed off. When a suitable substrate is added, the enzyme reacts with it to produce a color. This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample. The intensity of color/ optical density is measured at 450nm. The intensity of the color gives an indication of the amount of antigen or antibody.