Biofunctionalized semiconductor quantum dots for virus detection

Virus is a kind of microorganism and possesses simple structure and contains one nucleic acid,which must be replicated using the host cell system.It causes large-scale infectious diseases and poses serious threats to the health,social well-being,and economic conditions of millions of people worldwide.Therefore,there is an urgent need to develop novel strategies for accurate diagnosis of virus infection to prevent disease transmission.Quantum dots(QDs)are typical fluorescence nanomaterials with high quantum yield,broad absorbance range,narrow and size-dependent emission,and good stability.QDs-based nanotechnology has been found to be effective method with rapid response,easy operation,high sensitivity,and good specificity,and has been widely applied for the detection of different viruses.However,until now,no systematic and critical review has been published on this important research area.Hence,in this review,we aim to provide a comprehensive coverage of various QDs-based virus detection methods.The fundamental investigations have been reviewed,including information related to the synthesis and biofunctionalization of QDs,QDs-based viral nucleic acid detection strategies,and QDs-based immunoassays.The challenges and perspectives regarding the potential application of QDs for virus detection is also discussed.

A mini review and hypothesis for coronavirus detection using photonics: surface enhanced Raman scattering and fluorescence resonance energy transfer

COVID-19 has devastated numerous nations around the world and has overburdened numerous healthcare systems,which has also caused the loss of livelihoods due to prolonged shutdowns and further led to a cascading effect on the global economy.COVID-19 infections have an incubation period of 2–7 days,but 40 to 45%of cases are asymptomatic or show mild to moderate respiratory symptoms after the period due to subclinical lung abnormalities,making it more likely to spread the pandemic disease.To restrict the spread of the virus,on-site diagnosis methods that are quicker,more precise,and easily accessible are required.Rapid Antigen Detection Tests and Polymerase Chain Reaction tests are currently the primary methods used to determine the presence of COVID-19 viruses.These tests are typically time-consuming,not accurate,and,more importantly,not available to everyone.Hence,in this review and hypothesis,we proposed equipment that employs the properties of photonics to improve the detection of COVID-19 viruses by taking the advantage of typical binding of coronavirus with angiotensin-converting enzyme 2(ACE2)receptors.This hypothetical model would combine Surface-Enhanced Raman Scattering(SERS)and Fluorescence Resonance Energy Transfer(FRET)to provide great flexibility,high sensitivities,and enhanced accessibility.

Plasmon-enhanced FRET biosensor based on Tm^(3+)/Er^(3+)co-doped core-shell upconversion nanoparticles for ultrasensitive virus detection

Outbreaks of infectious viruses offer a formidable challenge to public healthcare systems and early detection of viruses is essential for preventing virus propagation.In this work,an ultrasensitive plasmon-enhanced fluorescence resonance energy transfer(FRET)biosensor based on core-shell upconversion nanoparticle(csUCNP)and gold nanoparticle(AuNP)for accurate detection of SARS-CoV-2 viral RNA is presented.In this biodetection assay,the Tm^(3+)/Er^(3+)co-doped csUCNP NaGdF_(4):Yb/Tm@NaYF_(4):Yb/Er acts as an energy donor and AuNP serves as an energy acceptor.The upconversion emission of Tm^(3+)and the design of the core-shell structure led to a simultaneous surface plasmon effect of AuNP.The localized surface plasmon resonance(LSPR)arising from collective oscillations of free electrons significantly enhanced FRET efficiency between Er^(3+)and AuNP.The as-prepared biosensor obtained a limit of detection(LOD)as low as 750 aM,indicating that the integration of FRET and surface plasmon into one biodetection assay significantly boosted the sensitivity of the biosensor.In addition,samples extracted from clinical samples are also utilized to validate the effectiveness of the biosensor.Therefore,this innovative plasmon-enhanced FRET biosensor based on Tm^(3+)/Er^(3+)co-doped csUCNP may pave the way for rapid and accurate biodetection applications.

Two-dimensional material-based virus detection

Cost-effective, rapid, and accurate virus detection technologies play key roles in reducing viral transmission. Prompt and accurate virus detection enables timely treatment and effective quarantine of virus carrier, and therefore effectively reduces the possibility of large-scale spread. However, conventional virus detection techniques often suffer from slow response, high cost or sophisticated procedures. Recently, two-dimensional(2D) materials have been used as promising sensing platforms for the highperformance detection of a variety of chemical and biological substances. The unique properties of 2D materials, such as large specific area, active surface interaction with biomolecules and facile surface functionalization, provide advantages in developing novel virus detection technologies with fast response and high sensitivity. Furthermore, 2D materials possess versatile and tunable electronic, electrochemical and optical properties, making them ideal platforms to demonstrate conceptual sensing techniques and explore complex sensing mechanisms in next-generation biosensors. In this review, we first briefly summarize the virus detection techniques with an emphasis on the current efforts in fighting again COVID-19. Then, we introduce the preparation methods and properties of 2D materials utilized in biosensors, including graphene, transition metal dichalcogenides(TMDs) and other 2D materials. Furthermore, we discuss the working principles of various virus detection technologies based on emerging 2D materials, such as field-effect transistor-based virus detection, electrochemical virus detection, optical virus detection and other virus detection techniques. Then, we elaborate on the essential works in 2D material-based high-performance virus detection. Finally, our perspective on the challenges and future research direction in this field is discussed.

An immune local concentration based virus detection approach

Along with the evolution of computer viruses, the number of file samples that need to be analyzed has constantly increased. An automatic and robust tool is needed to classify the file samples quickly and efficiently. Inspired by the human immune system, we developed a local concentration based virus detection method, which connects a certain number of two-element local concentration vectors as a feature vector. In contrast to the existing data mining techniques, the new method does not remember exact file content for virus detection, but uses a non-signature paradigm, such that it can detect some previously unknown viruses and overcome the techniques like obfuscation to bypass signatures. This model first extracts the viral tendency of each fragment and identifies a set of statical structural detectors, and then uses an information-theoretic preprocessing to remove redundancy in the detectors’ set to generate ‘self’ and ‘nonself’ detector libraries. Finally, ‘self’ and ‘nonself’ local concentrations are constructed by using the libraries, to form a vector with an array of two elements of local concentrations for detecting viruses efficiently. Several standard data mining classifiers, including K -nearest neighbor (KNN), radial basis function (RBF) neural networks, and support vector machine (SVM), are leveraged to classify the local concentration vector as the feature of a benign or malicious program and to verify the effectiveness and robustness of this approach. Experimental results show that the proposed approach not only has a much faster speed, but also gives around 98% of accuracy.

Establishment of Reverse-transcription Loopmediated Isothermal Amplification Method for Detection of Wheat Streak Mosaic Virus

A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Detection and Analysis of Ebola Virus in Sierra Leone-China Friendship Biosafety Laboratory from March 11 to April 20, 2015

Ebola virus disease reemerged in Western Africa in 2014.Chinese Center for Disease Control and Prevention dispatched the first Ebola virus（EBOV）detection team to run newly established Sierra Leone-China Friendship Biological Safety Laboratory.The aims of study were to understand epidemiology,clinical manifestations and survival time of EBOV in patient＇s blood.A total of 913specimens were tested between March 11 and April20, 2015. EBOV positivity occurred in 7.37% of the blood and 0.53% in throat swabs.

GENE ENGINEERING EB VIRUS MEMBRANE ANTIGEN IN DETECTION OF MA-IgA ANTIBODY(COMPARISON WITH VCA-IgA AND EA-IgA ANTIBODIES)

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluo-rescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pretreatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.

Study on Virus-free Culture and Rapid Propagation Technology of Xiangshu Series Varieties of Sweet Potato

[Objective] Sweet potato virus disease had a significant harm to the yield and quality of sweet potato, directly causing the degradation of sweet potato vari- eties and even the harvest failure. Therefore, the detection and removal of sweet potato virus and the establishment of rapid propagation method of sweet potato is of great significance to ensure the stable inheritance of excellent characters of sweet potato, prevent the spread of sweet potato virus and develop sweet potato industry. [Method] With Xiangshu series varieties of sweet potato, Xiangshu 15 and Xiangshu 19 as the research materials, a virus-free culture program was established for meristem tip apex tissue culture of different cultivars, and a rapid propagation method was developed for virus-free seedlings. [Result｜ On the basis of analysis on seedling emergence rate, the optimal addition scheme of plant hormones in the MS culture medium of Xiangshu 15 was 6-BA 3.0 mg/L ＋ NAA 1.0 mg/L, and the opti- mal plant hormone addition scheme for Xiangshu 19 was 6-BA 2.0 mg/L ＋ NAA 0.67 mg/L Under the developed rapid propagation system, the annual reproductive coefficient was up to 49 152, far higher than that （20 000） in field. IConclusionl Based on the actual production, combined with the meristem tip apex tissue culture, a comprehensive prevention and control measure was put forward, which included virus detection, early warning, removal and virus-free seedlings breeding, tt was of great strategic significance to improve the yield and quality of high-quality sweet potato and ensure the healthy development of sweet potato industry in China.

Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay

A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV-containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.

Hepatitis associated with hepatitis B virus in broilers

Infection by hepatitis B virus （HBV） results in acute and chronic liver damages in humans. Liver products of broilers as a primary food consumed in our daily life have a close connection with public health. The prevalence of the virus in livers and serum of broilers is of great significance, owning to the potential transmission between chickens and humans. Liver tissues and serum samples were tested to investigate the prevalence of hepatitis B virus infection in slaughtered broilers, for expression of HBV antigens and antibodies. The distribution and positive rate of hepatitis B surface antigen （HBsAg）, hepatitis B core antigen （HBcAg） and hepatitis B e antigen （HBeAg） in liver samples were examined using immunohistochemistry. HBsAg was mainly located in the cytoplasm of hepatocytes with a positivity of 81.61% whereas HBeAg and HBcAg were primarily located in the nucleus of hepatocytes with a positivity of 40.13 and 49.10%, respectively. Enzyme-linked immunosorbent assay （ELISA） analysis of serum for HBV serological markers demonstrated a high prevalence of hepatiits B surface antibody （HBsAb, 54.91%） and hepatitis B core antibody （HBcAb, 27.68%）, whereas HBeAb, HBsAg and HBeAg were rarely detectable. Classic hepatitis pathological changes, including swollen hepatocytes, focal parenchymal necrosis, lymphocytic infiltration and hyperplasia of fibrous connective tissues were observed using histopathological analysis. Some of the liver samples were found positive for HBV DNA using nested PCR. Sequence comparison confirmed that all sequences shared 97.5-99.3% identity with human HBV strains. These results demonstrated the existence of HBV in livers and serums of broilers. Animals or animal products contaminated with HBV could raise an important public health concern over food safety and zoonotic risk.

Complete Genome Sequence Analysis of Guangdong Isolates of Cymbidium Mosaic Virus

[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.

A Universal Real-Time Fluorescence qPCR Method for Identifying Epidemic Strains of African Swine Fever Virus

Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false negatives, and benefitting early diagnosis.

不同症状类型苜蓿病毒病AMV病原检测及其寄主范围测定

为明确采自田间不同症状类型苜蓿病毒病病样中苜蓿花叶病毒(alfalfa mosaic virus,AMV)的带毒情况、症状表现与叶绿素含量的相关性及AMV的寄主范围,本试验通过田间调查采样、症状归类、丙酮乙醇混合液法和双抗体夹心酶联免疫吸附测定(DAS-ELISA)法对不同病样的叶绿素和AMV含量进行测定和检测,并对提纯至不同症状类型病样中AMV对9科32种植物的致病性进行测定。结果表明田间苜蓿病毒病病样有轻花叶、重花叶、叶片边缘褪绿黄化型和叶片畸形皱缩花叶矮化型4种症状类型,均带有AMV,且叶绿素含量、AMV含量与症状表现之间具有正相关性,症状表现最严重的叶片畸形皱缩花叶矮化型病样的叶绿素a、b、总叶绿素含量均比对照低58.00%以上,类胡萝卜素含量比对照高134.06%,AMV含量最高,为252.96 pg·mL^(-1);寄主范围测定表明AMV可侵染7科27种植物,对西葫芦致病性最强,症状表现为局部枯斑,30 d时AMV浓度为316.19 pg·mL^(-1)。