Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Semi-implantable device based on multiplexed microfilament electrode cluster for continuous monitoring of physiological ions

Modern medicine is increasingly interested in advanced sensors to detect and analyze biochemical indicators.Ion sensors based on potentiometric methods are a promising platform for monitoring physiological ions in biological subjects.Current semi-implantable devices are mainly based on single-parameter detection.Miniaturized semi-implantable electrodes for multiparameter sensing have more restrictions on the electrode size due to biocompatibility considerations,but reducing the electrode surface area could potentially limit electrode sensitivity.This study developed a semi-implantable device system comprising a multiplexed microfilament electrode cluster(MMEC)and a printed circuit board for real-time monitoring of intra-tissue K^(+),Ca^(2+),and Na^(+)concentrations.The electrode surface area was less important for the potentiometric sensing mechanism,suggesting the feasibility of using a tiny fiber-like electrode for potentiometric sensing.The MMEC device exhibited a broad linear response(K^(+):2–32 mmol/L;Ca^(2+):0.5–4 mmol/L;Na^(+):10–160 mmol/L),high sensitivity(about 20–45 mV/decade),temporal stability(>2weeks),and good selectivity(>80%)for the above ions.In vitro detection and in vivo subcutaneous and brain experiment results showed that the MMEC system exhibits good multi-ion monitoring performance in several complex environments.This work provides a platform for the continuous real-time monitoring of ion fluctuations in different situations and has implications for developing smart sensors to monitor human health.

Development and Validation of Multiplex One-Step Real-Time TaqManqRT-PCR Assays for Detection and Quantification of Arboviral Encephalitis Viruses

Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.

Advances in the Identification of Genetically Modified Rice with Real-time PCR and Multiplex PCR

In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.

Development of a duplex real-time PCR method for the detection of influenza C and D viruses

Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.

Development of a multiplex one-step real-time RT-PCR assay for the simultaneous detection of eight viruses associated with febrile rash illnesses

Fever and rash illnesses(FRIs)are a series of common diseaseswith fever and rashes as clinicalmanifestations,most of which are caused by viral infection.The rashes of FRIs are generally nonspecific;therefore it is difficult to identify FRIassociated viruses solely based on clinical symptoms.To achieve rapid and accurate identification of FRI pathogens,a multiplex one-step real-time reverse transcription-polymerase chain reaction(RT-PCR)assay was developed and evaluated in this study.Primers and probes were selected for the detection of measles virus(MeV),rubella virus(RV),human enterovirus(EV),varicella-zoster virus(VZV),dengue virus(DENV),human parvovirus B19(B19),Epstein-Barr virus(EBV),and human herpes virus 6(HHV-6),which cover the most common pathogenic viruses of FRIs.Detection of the eight FRI-associated viruses,which was divided into two groups/tubes,was simultaneously performed under universal optimized reaction conditions in multiplex one-step real-time RT-PCR assay.The multiplex realtime RT-PCR showed high sensitivity and specificity in detecting the eight FRI-associated viruses.The limits of detection(LODs)for the eight viruses were in the range of 47–177 copies/reaction,and no cross reactions for the eight FRIassociated viruses were found in the multiplex assay.In addition,the results of the multiplex real-time RT-PCR assay were consistent with the results of a monoplex real-time RT-PCR assay and sequencing for clinical specimens obtained from FRI patients.With its advantages of high efficiency and rapid and accurate diagnosis,multiplex real-time RT-PCR was very feasible for the early diagnosis of FRI pathogenic viruses and would be of great help for the proper treatment,monitoring,and initiation of preventive measures for FRI cases.

Real-time spatiotemporal division multiplexing electroholography for 1,200,000 object points using multiple-graphics processing unit cluster

Computationally, the calculation of computer-generated holograms is extremely expensive, and the image quality deteriorates when reconstructing three-dimensional(3 D) holographic video from a point-cloud model comprising a huge number of object points. To solve these problems, we implement herein a spatiotemporal division multiplexing method on a cluster system with 13 GPUs connected by a gigabit Ethernet network.A performance evaluation indicates that the proposed method can realize a real-time holographic video of a3 D object comprising ~1,200,000 object points. These results demonstrate a clear 3 D holographic video at32.7 frames per second reconstructed from a 3 D object comprising 1,064,462 object points.

Identified Bacteria and Virus in the Cerebrospinal Fluid of under Five Years Hospitalized Children for Clinical Meningitis at Panzi Hospital in the Eastern Part of DRC

Background: Meningitis remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo). Distinguishing children with bacterial meningitis from those with viral meningitis in the emergency department is sometimes difficult. Here we identified bacteria and virus in the cerebral spinal fluid (CSF) of children with meningitis. Material and Methods: This is a prospective, analytical study carried out in the Pediatrics department of Panzi Hospital in the South-Kivu province of DR Congo. Between April 2021 and March 2022, 150 of 251 collected CSF from children aged from 1 to 59 months hospitalised due to clinical meningitis at Panzi referral university hospital, Bukavu, Eastern DR Congo were sent to the Lancet laboratory for bacteria identification by a multiplex real-time PCR assay for detection of the most different viruses and bacterial species causing meningitis. Result: The used multiplex real-time PCR assay allowed us to identify germs in 24.7% of cases (37/150). We isolated bacteria in 25/37 (67.5%) cases, and viruses in 9/37 (24.3%) while virus and bacteria co-infection was detected in 3/37 (8.1%). The most frequently identified bacteria were Streptococcus pneumoniae 14/37 (37.8%) followed by Haemophilus influenzae 6/37 (16.2%). The main virus was cytomegalovirus 5/37 (3.5%). Despite the age, the most found bacterial are common in children from rural areas and unvaccinated children. Bacterial and virus co-infection were identified in 66.7% of children aged between 25 - 60 months, mainly among male children, and in all children from rural areas (100%). The overall case fatality rate was 30% and was very high among cases with co-infection CMV-Pneumococcal (66.7%), followed by Streptococcus pneumoniae (50%). Conclusion: Meningitis remains frequent among children aged from one to 59 months among Bukavu Infants. We noticed that, Children with co-infection with bacteria and viruses might need higher attention when having meningitis symptoms, as this could lead to fatal outcomes. The introduction of molecular techniques, such as multiplex real-time PCR, has the potential to improve diagnosis and patient outcomes.

Genetic Characteristics of Lipooligosaccharide and Capsular Polysaccharide of Campylobacter jejuni from Different Sources in China

Objective To determine the distribution of two important virulence factors[lipooligosaccharide(LOS)and capsular polysaccharide(CPS)]in Campylobacter jejuni(C.jejuni)isolated from different sources in China and to develop a rapid screening method for Guillain–Barrésyndrome(GBS)-associated strains.Methods Whole-genome sequencing was carried out for 494 C.jejuni strains.The Ortho MCL software was used to define the LOS/CPS gene clusters.CPS genotyping was performed with serotype-specific sequence alignment using the BLAST software.Real-time Polymerase chain reaction(PCR)was developed with the unique sequences of specific CPS types.Results Nine novel and 29 previously confirmed LOS classes were identified.LOS classes A,B,and C were the most common(48.2%,238/494)among the 494 strains.Twenty-six capsular types were identified in 448 strains.HS2,HS4c,HS5/31,HS19,and HS8/17 were the most frequent CPS genotypes(58.7%,263/448).Strains of 17 CPS genotypes(strain number>5)had one or two prevalent LOS classes(P<0.05).Multiplex real-time PCR for rapid identification of HS2,HS19,and HS41 was developed and validated with strains of known serotypes.Conclusion Our results describe the genetic characteristics of the important virulence factors in C.jejuni strains in China.The multiplex real-time PCR developed in this study will facilitate enhanced surveillance of GBS-associated strains in China.

Diagnostic assays developed for the control of foot-andmouth disease in India

Foot-and-mouth disease(FMD) is a highly contagious and economically devastating disease of livestock, primarily affecting cattle, buffalo and pigs. FMD virus serotypes O, A and Asia1 are prevalent in India and systematic efforts are on to control and eventually eradicate the disease from the country. FMD epidemiology is complex due to factors like co-circulation, extinction, emergence and re-emergence of genotypes/lineages within the three serotypes, animal movement, diverse farm practices and large number of susceptible livestock in the country. Systematic vaccination, prompt diagnosis, strict biosecurity measures, and regular monitoring of vaccinal immunity and surveillance of virus circulation are indispensible features for the effective implementation of the control measures. Availability of suitable companion diagnostic tests is very important in this endeavour. In this review, the diagnostic assays developed and validated in India and their contribution in FMD control programme is presented.

一种快速获取港口控制点坐标的方法

目前港口控制点坐标主要采用全球导航卫星系统(global navigation satellite system,GNSS)静态观测方式获取。介绍了实时扩展后处理(real-time extended post-processing,RTX-PP)技术服务,结合港口基础控制测量案例,利用RTX-PP技术获取控制点国际地球参考框架(international terrestrial reference frame,ITRF)2014的2010.0历元坐标,采用平面四参数坐标转换方法求取上述坐标与2000国家大地坐标系(China Geodetic Coordinate System 2000,CGCS2000)坐标的转换参数,内检核点位精度为0.007 m,外检核点位精度为0.009 m。通过工程应用对方法进行验证,与常规GNSS静态解算成果相比,RTX-PP转换成果点位精度约0.029 m,差值主要由区域速度场差异造成。利用RTXPP技术可快速获取港口高等级控制点的CGCS2000坐标。

A rectangle bin packing optimization approach to the signal scheduling problem in the Flex Ray static segment

As FlexRay communication protocol is extensively used in distributed real-time applications on vehicles, signal scheduling in Flex Ray network becomes a critical issue to ensure the safe and efficient operation of time-critical applications. In this study, we propose a rectangle bin packing optimization approach to schedule communication signals with timing constraints into the FlexRay static segment at minimum bandwidth cost. The proposed approach, which is based on integer linear programming(ILP), supports both the slot assignment mechanisms provided by the latest version of the FlexRay specification, namely, the single sender slot multiplexing, and multiple sender slot multiplexing mechanisms. Extensive experiments on a synthetic and an automotive X-by-wire system case study demonstrate that the proposed approach has a well optimized performance.