Detection Of Zaire Ebola Virus By Reverse Transcription–loop-mediated Isothermal Amplification

Ebola virus disease(EVD),formerly known as Ebola haemorrhagic fever)is a severe and usually fatal illness with a death rate of up to 90%,which is caused by Ebola virus,a member of the filo-virus family.EVD first appeared in 1976 and caused two simultaneous outbreaks,it occurred in a village near the Ebola River,from which the disease takes its name.Zaire Ebola virus caused an outbreak in 2014 in West Africa,which was the most serious international Public Health Emergency since SARS.WHO concluded that this outbreak was the largest and most complex one since the Ebola virus first discovered in 1976.There have been more cases and deaths during this outbreak than all others combined.It has also spread between countries,starting in Guinea and then spreading across land borders to Sierra Leone and Liberia,by air to Nigeria and USA,and by land to Senegal and Mali.The epidemic in 9countries such as West Africa,Europe and America caused a total of more than 29000 people infected and 11000 people died,with the mortality of 39.7%.Furthermore,this outbreak caused huge economic losses of 320 billions of dollars and international social panic.The United States,Britain,France and other countries,several aid agencies and international organizations have invested enormous human and material resources to combat the epidemic in West Africa.China has sent a number of rescue forces including P3 mobile laboratory team,fixed laboratory team and the medical team to keep the epidemic outside our country and help the infected countries and people.The Ebola virus incubation period is 2-21 days.It is difficult to distinguish EVD from other infectious diseases such as malaria,typhoid fever and meningitis.To confirm the symptoms caused by Ebola virus infection,the following methods are available:antibody-capture enzyme-linked immunosorbent assay(ELISA),antigen-capture detection tests serum neutralization test,reverse transcriptase polymerase chain reaction(RT-PCR)assay,electron microscopy and virus isolation by cell culture.The most severely affected countries,have very weak health systems and lack human and infrastructural resources.The demand for high precision instruments and equipment of existing detection method is difficult to realize,thus it is very important to establish a simple,easy operating and fast detection method.Loop-mediated isothermal amplification(LAMP)is a one-step nucleic acid detection method developed in 2000.Since 2000,LAMP has become a hot research in the field of nucleic acid detection for its property advantages of rapidity,simple and unnecessary precise instrument and so on.And then LAMP have been received extensive attention in academic circles,WHO and the relevant government departments.Just a few years,LAMP assays have been widely applied to the detection of SARS,avian influenza and HIV disease.The reaction principles of LAMP are as follow: this method employs a set of six specially designed primers that recognize a total of six distinct sequences on the target DNA with the help of a Bst DNA polymerase starting strand displacement reaction.A large number of target DNA production are monitored by magnesium pyrophosphate formation.This feature can make the LAMP reaction processes been judged negative or positive directly by monitoring turbidity of the reaction.Because LAMP recognizes the target by six distinct sequences initially and by six distinct sequences afterwards,it is expected to amplify the target sequence with high selectivity.LAMP amplification process can be carried out under constant temperature conditions,a stable heat source equipment will be able to satisfy the requirements,no need for precised instruments.Reverse Transcription Loop-mediated isothermal amplification(RT-LAMP)is one of LAMP technology for RNA virus.In addition to LAMP reaction system,RT-LAMP increase the reverse transcriptase.Combination of Reverse transcription and nucleic acid amplification greatly reduces the time for virus detection.Generally,LAMP can be used for the poor health conditions of the West African,and also be used for the lack of professional and technical personnel and the detection of the heavy task of China's port quarantine agency.The virus causing the 2014 West African outbreak belongs to the Zaire species.The Ebola virus is only prevalent in Africa currently,but does not rule out the possibility of its spread to other continents,thus rapid detection of Zaire ebola virus is very important.The ebola virus has a high mortality rate.Samples from patients are an extreme biohazard risk.Laboratory testing on non-inactivated samples should be conducted under absolutely biological non-containment conditions.So we constructed the fake virus based on the NP protein of Zaire ebola virus as a reference standard of the virus.It can replace Zaire ebola virus as detecting targets.Our aim was to establish a good nucleic acid detection method for Zaire ebola virus.NP,a highly conserved gene among all Ebola virus species,plays an important role in the replication of the viral genome and is essential for formation of the nucleocapsid.Five sets of primers were designed targeting the published NPsequences of Zaire ebola virus.Compared the five sets of primers amplification efficiency to get the best primer combinations,and optimize the primers concentration ratio.Two methods,namely,real-time monitoring of turbidity and addition of calcein to the reaction tube,were used to determine the LAMP results.The results showed that target DNA were amplified and visualized by the two detection methods within50 min at an isothermal temperature of 61?.The sensitivity of LAMP,with a detection limit of 4.56 copies/min at an isothermal temperature of 61 oC were used to determine LAMP results.The results showed that the LAMP method has high specificity.Consequently,the LAMP described here has the potential to become a useful tool for the rapid detection of Zaire ebola virus in the situation of poor-resource and time-aspired,especially point of care.The test results for simulating clinical samples show that the method has a good stability.Based on the method of Zaire Ebola detection,we cooperated with the Beijing aipuyi biotechnology company to develop Diagnostic Kit for Zaire ebola virus RNA(RT-LAMP),which rapidly detected Zaire Ebola virus quantificational from the blood or throat swab specimens in RNA.The Kit was mainly used for qualitative detection of Zaire type Ebola virus in blood or throat swab specimen RNA,which is favorable for the auxiliary diagnosis and epidemiological detection of the virus.The sensitivity of the Kit is high,with detection limit of 1 × 103 copies/ml.The kit has good stability when freeze-thaw is less than 6 times and can be used for long-distance transportation.China's assistance to Sierra Leone laboratory second batch testing team used it as a way of clinical samples in the early screening in Sierra Leone.All clinical samples were analyzed by RT-LAMP and real-time RT-PCR simultaneously.The results showed that 307 patients were confirmed cases of ZEBOV infections and 106 patients were tested as negative for EBOV by RT-LAMP,the sensitivity and specificity were agreed with the results of RT-PCR.We developed and optimized a novel RT-LAMP assay specific for ZEBOV diagnosis using primers spanning the 663 bp NP sequence of the viral genome.Our data showed the RT-LAMP assay developed in this study is rapid,simple,highly specific,and sensitive for the detection of ZEBOV.It can be used for the Zaire Ebola virus disease screening,for diagnosing positive cases as soon as possible and making relevant measures to control the epidemic,so that we can better scale and reduce the losses caused by the epidemic,and ultimately improve the ability to respond to this new,unexpected infectious disease.