Even though restrictions are beginning to ease up in certain countries, COVID-19 continues to pose a severe and urgent threat to global public health. Selecting appropriate detection techniques and methods to make accurate and rapid identification of etiology plays a key role in improving the efficiency of diagnosis and therapy, as well as restraining the spread of the disease. Researchers have developed several rapid and accurate diagnostic tests for coronavirus SARS-CoV-2 infection, including metagenomic sequencing, real-time quantitative fluorescent PCR (qRT-PCR), isothermal amplification technologies, CRISPR/Cas gene editing technology, serum/plasma detection of IgM/IgG antibodies, etc.
- Metagenomics Sequencing
The advantage of viral metagenomic sequencing (mNGS) is that it can screen unknown infection sources in the early stages of an epidemic and provide evidence of virus mutation to help to control of the epidemic. In this method, viral RNA is extracted from patient samples, a viral cDNA library is constructed, then high-throughput sequencing and database comparison are performed to identify infection sources and analyze the strain’s homology and evolution.Metagenomics Sequencing Characteristics:
a. Highly accurate.
b. Simultaneously detect a variety of pathogenic microorganisms, including bacteria, fungi, viruses, and parasites, while comprehensively monitoring the potential risk of infectious diseases.
c. High requirements for sequencer and analysis data calculation systems, as well as high technical requirements of operators.
d. Long detection turnaround time with an average detection time of about 48h or longer.
qRT-PCR technology is used to isolate viral RNA from tissues, cells, body fluids, and other types of samples, to then use specific primers to amplify the viral genes by RT-PCR reaction. Finally, the RNA is quantified by the strength of its fluorescence signal. In general, it’s the first clinically available way of mass population screening. Based on the complete genome sequence of SARS-CoV-2, three specific regions of novel Coronavirus (ORF1ab, N, and E) are recommended as suitable for PCR amplification.qRT PCR Characteristics:
a. The sensitivity of the method is high enough to achieve less than 10 copies/reaction.
b. Main method for early diagnosis of the epidemic (development of antigens and antibodies takes time).
c. False negative results exist (CT imaging diagnosis is positive, nucleic acid is negative), and the possible reason is an off-target primer caused by mutation, nasopharyngeal swab sampling problems, or sample transportation problems.
The RT-LAMP method can design specific primers for multiple regions of target genes and can use chain substitution DNA polymerase for loop-mediated isothermal amplification so that the target sequence can be amplified exponentially. LAMP can be completed in less than 1 hour at 60~65°C without the use of thermal circulation instruments.
a. High sensitivity and specificity.
b. Isothermal amplification at 60-65 °C.
c. Visual detection can be performed by colorimetric, turbidimetric, or fluorescent dye methods.
Like LAMP, RPA is also an isothermal amplification method. However, the reaction temperature of RPA is lower than that of the LAMP method, thus the reaction can be completed at room temperature without external heating. This method can be combined with probes for real-time fluorescence quantification or adding dye for visual detection, which is more suitable for rapid and real-time detection.RT-RPA Characteristics:
a. Higher sensitivity, and faster reaction speed.
b. Amplification at room temperature.
c. The patentability of related reagents and high cost limit the application of this method in real-time detection.
- CRISPR/Cas System
The CRISPR/Cas system is a common tool for gene editing, which has the ability to accurately recognize and cleave specific sequences. Activated Cas13a and Cas12a enzymes can not only cleave a targeted sequence, but also can cleave non-specific RNA or DNA probes in the surrounding environment to realize sensitive and specific nucleic acid detection.CRISPR/Cas System Characteristics:
a. High sensitivity and can conduct high-throughput detection.
b. Combined with isothermal amplification technology, multi-target rapid detection can be realized.
- IgM/IgG Antibody Detection
IgM and IgG are specific antibodies that appear successively in the human body after viral infection. Detection methods based on IgM and IgG double antibodies have advantages of high sensitivity, diagnosis timeliness, and flexible and extensive application. Detection methods in this category mainly include colloidal gold method, ELISA, and chemiluminescence methods. These three methods can detect total IgM/IgG antibodies or IgM and IgG antibodies respectively.IgM/IgG Antibody Detection Characteristics:
a. The operation method is simple and the requirement of instrument is low.
b. The detection time is short and the sensitivity is high.
c. The high false positive rate may be caused by hemolysis of samples, fibrin, bacterial contamination, or autoantibodies of patients.
RT-PCR nucleic acid detection + serum antibody detection is the main method for the diagnosis of COVID-19. Oligonucleotides (the specific primers, diagnostic probes, fluorescent probe, etc) are the core materials of nucleic acid detection kits. The oligonucleotide’s purity, mutation rate, and any other factors play a great effect of the experiment result. Therefore, the selection of appropriate oligonucleotide synthesis and purification methods are also the important basis of an experiment.
All oligonucleotides from Synbio Technologies are manufactured in compliance with ISO 9001:2015 & ISO 13485:2016 quality management system standards. Through strict raw material supplier screening, instrument performance testing, mass DMT testing, purity analysis, mutation rate monitoring, and other rigorous intermediate process controls, our average oligo synthesis coupling efficiency is more than 99% in order to ensure the high quality and purity of our products.
Let’s take a look at how the oligos are made