The genomic sequences of most viruses have been known. Nucleic acid probes which are short segments of DNA designed to hybridize with complementary viral DNA or RNA segments . The polymerase chain reaction (PCR) is a more efficient technique for viral detection. High throughput diagnostic methods have been developed recently.
A. Nucleic acid hybridization technique
Nucleic acid hybridization, mainly including Southern blotting(Southern) and Northern blotting(Northern), is a fast developing new technique in the virus diagnostic field. The rationale of the hybridization assay is to use short segments of DNA(called “probe”) designed to hybridize with complementary viral DNA or RNA segments. By heating or alkaline treatment, double-stranded target DNA or RNA are separated into single strands and then are immobilized on a solid support. After that, probe is added and hybridized with the target DNA or RNA. As the probe is labeled with isotope or non-radioactive nuclide, the target DNA or RNA can be detected through autoradiography or by the biotin-avidin system . Since most of viral genomes have been cloned and sequenced, they can be detected using virus–specific sequences as probes in the specimen. Currently, the hybridization methods includes: dot blot , in situ hybridization in cells , DNA blotting(DNA) (Southern blot) and RNA blotting(RNA) (Northern blot).
In recent years, a series of in vitro nucleic acid amplification techniques has been developed based on PCR, to test insensitive or uncultivable viruses. PCR is a method which can synthesis specific DNA sequence by in vitro polymerase reaction. The process of PCR includes a thermal cycle of three steps: denaturation , annealing , and extension At high temperature (93℃~95℃), the double-stranded DNA is separated into two single DNA strands; then at low temperature (37℃~60℃), two synthesized nucleotide primers anneal to the complementary DNA segments; whereas at the appropriate temperature for Taq enzyme (72℃), synthesis of new DNA chains start from primer 3’end using complementary DNA as templates and single nucleotides as materials. So after each cycle, one DNA chain can be amplified into two chains. Repeating this process, each DNA chain synthesized in one cycle can be used as template in the next cycle, and the number of DNA chains is doubled in each cycle, which means the production of PCR is amplified in a 2n log speed. After 25 to 30 cycles, the production of PCR is identified through electrophoresis , and the specific DNA products can be observed under UV light (254nm). For its advantage of specificity, sensitivity, and convenience, PCR has been adopted in clinical diagnosis of many viral infections such as HCV, HIV, CMV, and HPV. As PCR is very sensitive，it can detect virus DNA at fg level, the operation should be carried out very carefully to avoid false positive. In addition, positive result in nucleic acid test doesn’t mean there is live infectious virus in the sample.
With wide application of PCR technique, new techniques and methods are developed based on PCR technique for different test purpose. For instance, the real time quantitative PCR can detect viral load; in situ PCR is used to identify virus infection in tissue or cells; The nested PCR can increase the specificity of PCR. Among them, real time quantitative PCR has been developed more rapidly. Many new techniques, such as TaqMan hydrolysis probe, hybridization probe, and molecular beacon probe, have been combined into real time quantitative PCR technique, which is widely utilized in clinical research. Besides identifying the viral load in patients’ body fluid accurately, this method can also be used to detect drug-tolerant mutant. Therefore, real time quantitative PCR is mainly applied in curative effect evaluation and drug tolerance surveillance.
C. High-throughput detection of viral nucleic acids
To meet the needs for fast diagnosis of new emergent infectious diseases, various high-throughput detection methods, like DNA chips(DNA), have been established. For DNA chips, specific probes are synthesized and attached to small silicon chips in very high density to form DNA probe microarray (DNA) which can be hybridized with sample. The signal of hybridization can be imaged by confocal microscope or laser scanner and be further processed by the computer and huge data set concerning different genes can be obtained. There are two kinds of DNA chip. The “synthesis chip” is as following: the specific oligonucleotides are synthesized directly on the chips. Another is DNA pool chip. The cloned genes or PCR products are orderly printed on the slide. The advantage of DNA chip technology is the simultaneous detection of a huge quantity of DNA sequences. The latest version of pathogen detection chip can identify over 1700 human viruses at once. DNA chip technology solved the problems of traditional nucleic acid hybridization methods and has very broad applications in viral diagnosis and epidemiological study.