How is Ebola detected?

According to media reports, Shenzhen BGI And the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences have successfully developed a nucleic acid detection reagent for Ebola virus, and have applied for emergency approval from the State Food and Drug Administration. 1

If the approval is approved and it enters clinical application as soon as possible, it will greatly enhance China's response to the Ebola virus. At present, commonly used test methods for viral infection include isolation and identification of viruses, serological diagnosis of viruses, and detection of viral components (protein antigens and nucleic acids).

Collection and submission of specimens

Specimens used to isolate the virus or to detect the virus and its nucleic acids should be taken from the patient's acute phase. Specimens from different sites (e.g., nasopharyngeal discharge, cerebrospinal fluid, feces, or blood) are taken depending on the viral infection. And pay attention to the use of antibiotics to inhibit the growth and reproduction of bacteria or fungi in the specimen. Since viruses are easily inactivated at room temperature, care should be taken to refrigerate specimens in the collection and transport of specimens.

For the taking of serological diagnostic specimens, one serum should be taken at the beginning of the disease and within 2 to 3 weeks after the disease to facilitate the dynamic observation of the titer of the double serum antibody.

Isolation and identification of viruses

Because the virus has strict intracellular parasiticity, the corresponding cell tissue, chicken embryo or sensitive animal should be selected for virus isolation and identification according to the type of virus, which is the gold standard for viral pathogenic diagnosis. However, the method is complex and requires a long time, so it is not suitable for clinical diagnosis, and is only suitable for laboratory research or epidemiological investigation of the virus.

Animal inoculation is the earliest method of virus isolation and is not used much at present. According to the affinity of the virus, the inoculation site of the sensitive animal can be selected, and the incidence of the animal can be observed and serological tests can be performed. The method is simple, the experimental results are easy to observe, and the method is still used for some cells that have been cultured in the morning. However, animals are not sensitive to many human viruses, or the symptoms are not obvious after infection, and animals often carry potential viruses, and these potential viruses should be prevented from being mistaken for inoculated pathogens.

Chicken embryos are sensitive to a variety of viruses, and chicken embryos are usually used to incubate for 9 to 14 days. Because chicken embryos are the most sensitive to influenza viruses, in addition to the isolation of influenza viruses, the isolation of other viruses has basically been replaced by cell culture.

Cell culture is the most commonly used method for virus isolation and identification. It can be divided into monolayer cell culture and suspension cell culture according to the way the cells are grown. From the source of the cells. Chromosomal characteristics and passage times can be divided into primary cells, diploid cells, passage cell lines and so on.

Morphological identification of viruses, i.e. morphological observation and determination of size by electron microscopy and immune microscopy.

Serological identification of viruses, that is, serological identification of viruses by known antibodies of species, types and subtypes, in addition to the commonly used immunomarking methods, there are blood coagulation suppression tests.

The main methods for the molecular biology identification of viruses include nucleic acid amplification, nucleic acid hybridization, gene chips, gene sequencing and other molecular biology techniques.

For viruses with few serotypes or viruses that do not yet proliferate successfully in ordinary cell culture systems, direct antigen detection is a quick and practical approach. This method requires a certain amount of antigen and high-quality antibodies in the specimen; The principle is immunological technique, i.e., the detection of the corresponding antigen with specifically labeled antibodies. Antigens in locally shedding or secreting cells infected with viruses can be detected with immunofluorescence or immunase-labeled antibodies, or antigens can be detected by enzyme-linked immunoassay (elisa) or latex agglutination. Results can be obtained in a few hours or within a day.

IGM antibodies in the serum of virally infected people can be detected with specific antigens to quickly diagnose the pathogen. The applied viral antigen or a recombinant antigen expressed using genetic engineering, or a synthetic peptide derived from the coding gene fragment may also be used as an antigen. Since imm antibodies appear early in viral infection, the timing of specimen collection has a great influence on the detection results of this method. In addition, the quality of the antigens used and the magnitude of the coverage of the epitopes of the antigens also affect the test results. Viral proteins can also be electrophoretically passed by gel and then transferred to membranes, and antibodies to a certain viral antigen subunit in the serum can be detected by a method of staining with a serum specimen (called western blotting, western blotting, or western blotting).

Since most viral genes have been successfully cloned and sequenced, viral genes can be used as probes to detect the presence of corresponding viral nucleic acids in specimens by nucleic acid hybridization. Viral nucleic acids that act as probes can be labeled with isotopes or non-radionuclides. After hybridization with probes, nucleic acid hybrids can be detected by autoradiography or with biotin-avidin systems. This method is generally not highly sensitive, but it is useful when the specimen contains a large amount of viral nucleic acid. After using gel electrophoresis to electrophoresis, the DNA in the specimen is transferred to the membrane (southen blotting method), and then the viral probe is used as a nucleic acid hybridization, which can distinguish the presence of viral nucleic acids in the specimen according to the size of the molecular weight, such as integration type or free type.

For viruses in which the nucleotide sequence of the gene has been determined, primers of the corresponding viral gene can be designed for polymerase chain reaction (PCR). The principle is to first add the nucleic acid extracted from the specimen (according to the virus to be tested as rna virus or DNA virus and add rna or DNA), and the RNA needs to be transcribed into complementary DNA first, and after adding heat-resistant DNA polyase, it is PCR at a certain temperature and conditions. Amplification of viral gene fragments can diagnose the presence of viral nucleic acids in specimens. This method is very sensitive, but it is necessary to pay attention to the false positives that occur from contamination during operation.

The disadvantage of detecting viral nucleic acids is that a positive viral nucleic acid does not mean that an infectious live virus is present in the specimen. In addition, for unknown viruses and new viruses that may appear, these methods cannot be used because the nucleotide sequence of the virus is not understood.

Due to the long cell culture cycle of the virus and the need for Ebola virus isolation in bsl-4 laboratories, there is currently no laboratory in China that meets this biosafety level; Low sensitivity of serological methods; Molecular biology methods include rt-pcr, real-time fluorescence rt-pcr, nested PCR, and ring-mediated isothermal amplification techniques. 2

It is reported that the reagents sent for examination and testing this time are precisely the application of molecular biology methods. Regarding the specific detection process, first of all, the researchers emphasized that for suspicious patient samples (blood and other body fluids), nucleic acid extraction should be completed in a biosafety level 3 or above laboratory, or 60 degree inactivation of nucleic acids for detection after 1 hour, the reagent is through fluorescent polymerase chain reaction (PCR) technology, the entire detection time takes about 3 to 4 hours.

bibliography

1. Fruit Shell Network, China's Ebola Detection Reagent Application For Emergency Approval, 2014.8.14, http://www.guokr.com/article/438996/

2. Xia Wenying, Zheng Kui, Huang Jicheng, et al., A Preliminary Study on the Preparation of Gene Chips for Ebola Virus Detection[j], Chinese Journal of Border Health and Quarantine, 2011, 34(3): 150-154