Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.     

The partial purified RNA-dependent RNA polymerases from bamboo mosaic potexvirus and potato virus X infected plants containing the template-dependent activities

RNA-dependent RNA polymerases (RdRp) isolated from bamboo mosaic potexvirus (BaMV) and potato virus X infected Nicotiana benthamiana plants and solubilized with the detergent NP-40, generated a full-length genomic and two subgenomic double-stranded RNAs of respective viruses in an in vitro RdRp assay containing endogenous RNA templates. Template-dependent and species-specific RdRp activity could be detected after the removal of endogenous RNA templates. The 3′ untranslated regions (UTR) containing a stretch of 40 adenylate residues were shown to be an efficient exogenous RNA template for in vitro RdRp reactions. Solution hybridization and nuclease digestion studies revealed that the products transcribed in vitro were minus-sense. Besides using the 3′ UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3′ terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis. Promoter studies with BaMV RdRp showed that domain D containing the potexviral hexamer motif of the 3′ UTR would be the major contributor of minus-sense RNA synthesis in vitro. On the other hand, the pseudoknot domain containing the poly(A) sequences would be sufficient for minus-sense RNA synthesis.     

Development and evaluation of one‐step multiplex real‐time RT‐PCR assay for simultaneous detection of Zika virus and Chikungunya virus

Zika virus (ZIKV) and chikungunya virus (CHIKV) are important human pathogens and mosquito‐borne arboviruses, which have resembling history, common vectors, circulating regions, and indistinguishable clinical symptoms. Wide geographical range that is suitable for ZIKV and CHIKV transmission underlines the concern about the impact of epidemic and endemic infections on burden of public health. In the present study, a highly sensitive and specific one‐step multiplex real‐time RT‐PCR assay was developed and evaluated for simultaneous detection and quantification of ZIKV and CHIKV. The single reaction assay employs two pairs of primers and two TaqMan probes that differentiate ZIKV and CHIKV infections. The entire viral genomic RNA in vitro transcribed from full‐length infectious clones were used to generate the standard curves for absolute quantification in subsequent tests. The detection limit of the one‐step multiplex assay was 1 and 0.5 PFU for infectious ZIKV and CHIKV, respectively. The assessment of specificity indicated this assay is highly specific to targeted viruses showing no amplification of a variety of other flaviviruses. Our assay was able to detect geographically separated and phylogenetically diverse strains of ZIKV and CHIKV. On the applicability of monitoring viral multiplication in cells and testing clinical samples, the one‐step multiplex assay provided efficient and accurate determination. The one‐step multiplex real‐time RT‐PCR assay offers a valuable tool for detection of ZIKV and CHIKV and potentially contributes to general surveillance and clinical treatment.

     

Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).     

Development of a multiplex bead-based assay for detection of hepatitis C virus

Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting approximately 3% of the human population. Although HCV detection is currently based on reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate, and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5, and Combined, we describe a new bead-based multiplex test capable of detecting HCV infection in human serum samples. The first analysis, made in a singleplex format, showed that each antigen coupled to an individual bead set presented high-level responses for anti-HCV-positive reference serum pools and lower-level responses for the HCV-negative pools. Our next approach was to determine the sensitivity and specificity of each antigen by testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format, the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and 55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%. All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex assay, which resulted in increased sensitivity and specificity values, performing with 100% sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex assay were 100%. Although preliminary, this 4-plex assay showed robust results that, aligned with its small-sample-volume requirements and also its cost- and time-effectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.     

Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high-risk populations. The aim of this study was to develop a real-time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID50 of virus stock. Three hundred eighty-nine clinical samples were tested in total from two patient groups. Overall, the real-time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real-time influenza A assay demonstrated clinical usefulness in both hospital and community populations.     

Development and validation of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs

A simplified, cost-effective, two-step duplex quantitative real-time RT-PCR assay was shown to detect and quantify foot-and-mouth disease virus positive-stranded RNAs and negative-stranded RNAs simultaneously for improved investigation of the state of virus infection and replication. The primers and Taqman probes were selected from the coding regions of 2B gene and 3D gene respectively, which have the least variations among serotypes. Cells infected acutely, tissue samples and single cell samples were used for evaluation of the assay. At the early stages of virus infection in vitro, the replication level reached a peak at 9 h.p.i. and the negative strands were detectable until 3 h.p.i. The kinetics of ratios of positive strands to negative strands (+RNA/−RNA) in vivo in the liver, kidney and spleen were similar, which demonstrated that the replication dynamics were similar in the three organs. 55 single cell samples out of 187 were positive by both positive strands qPCR and negative strands qPCR, the ratios (+RNA/−RNA) ranged from 15.6 to 1463.4 which showed considerable difference among single cell samples, indicating that active viral replication differs greatly in single cells. A duplex quantitative real-time RT-PCR was validated as effective and reliable.     

A novel virus of the late blight pathogen, Phytophthora infestans, with two RNA segments and a supergroup 1 RNA-dependent RNA polymerase

Double-stranded RNA representing four distinct electrophoretic patterns was found in a screen of Phytophthora infestans isolates. Two dsRNAs that always appeared together were sequenced. RNA 1, which was 3160 nt plus a poly (A) tail, contained a single deduced ORF with the potential to encode a polyprotein of 977 aa with motifs characteristic of supergroup I viral RdRps. The 2776 nt, polyadenylated RNA2 contained an ORF with a potential to encode a polyprotein of 847 aa including a possible trypsin-like serine protease, and a second putative ORF of unknown function. An alternative form of RNA2, in which a 19-nt stretch was replaced by a 9-nt sequence, was detected in 4 of 17 clones sequenced. Based on genome structure and phylogenetic analysis, this virus did not fit into any known virus family and we tentatively named it Phytophthora infestans RNA virus 1 (PiRV-1).     

登革病毒悬液芯片分型检测方法的建立

目的 建立一种基于悬液芯片的登革病毒(dengue virus,DV)检测方法,可对四种血清型登革病毒进行快速检测和鉴定.方法 依据GenBank上4种病毒的基因序列信息,设计并合成相关引物及探针序列.抽提病毒RNA,经反转录后对目的基因进行PCR扩增,产物与核酸探针微球组杂交后于Bio-PlexTM 200系统检测荧光信号值.结果 DV1的悬液芯片检测敏感性约9 DNA拷贝,DV2、DV3、DV4的悬液芯片检测敏感性约90 DNA拷贝.进而将本方法用于检测15份临床标本,其检测结果与分型荧光RT-PCR一致.结论 建立了可同时检测四种血清型登革病毒的悬液芯片检测方法,为快速筛查和鉴定登革病毒提供了新的手段.     

Development and evaluation of a real-time PCR assay for rapid identification and semi-quantitation of measles virus

A real-time PCR assay for measles virus was designed and validated using clinical samples including oral fluids, sera, urines, throat swabs, blood samples, and nasopharyngeal aspirates. The test was specific for measles virus, with a slightly higher sensitivity compared to the conventional nested PCR. Calculation of viral genome number in these samples, by comparison with a standard curve prepared from dilutions of cloned measles virus H gene, indicated that, overall, serum samples tended to have a lower viral load than oral fluid samples, and that the viral load decreased with increasing time after onset of symptoms. The real-time PCR is considered to be a sensitive and specific alternative to the conventional measles PCR, especially in situations where early and rapid diagnosis are important.     

Development and evaluation of a panel of multiplex one-tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions

Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses.     

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.     

Quantitative multiplex assay for simultaneous detection of the Indiana serotype of vesicular stomatitis virus and HIV gag

Assessment of in vivo viral replication of live attenuated recombinant vesicular stomatitis virus (rVSV) vaccine vector candidates encoding HIV gag requires comprehensive preclinical safety studies, and development of sensitive assays to monitor the outcome of vaccination of animals is important. In this study, two 2-step quantitative real-time RT-PCR assays were developed; a singleplex assay to detect VSV genomic RNA from ferrets inoculated intra-cranially (IC) or intra-nasally (IN) with either a wild-type (wt) virus or an attenuated rVSV vector engineered to express HIV gag protein, and a duplex assay to simultaneously detect VSV-N and HIV-gag mRNAs from cynomolgus macaques inoculated intra-thalamically (IT) with the same viruses. Using synthetic oligonucleotides as standards, the lower limit of detection of VSV-N and HIV-gag was 50 copies. Results showed high levels of wt VSV(IN) genomic RNA and mRNA in ferret and macaque tissues, respectively, and significantly lower levels of VSV genomic RNA and VSV-N and HIV-gag mRNAs in tissues from animals inoculated with the attenuated rVSV vector. These assays correlated with both the course of infection for these animals, and the infectious viral load measured by a standard plaque assay, and could be used to determine the safety profile of rVSV vaccine vectors.     

Development of a multiplex single base extension assay for mitochondrial DNA haplogroup typing

Aim

To provide a screening tool to reduce time and sample consumption when attempting mtDNA haplogroup typing.

Methods

A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 21 samples inconclusively haplogroup typed by CR data, 20 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex.

Results

When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II.

Conclusion

The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies.Mitochondrial DNA (mtDNA) is a 16 569 bp circular molecule present, on average, in 500 copies per cell (1). MtDNA analysis is utilized in several areas of science including, but not limited to, anthropology, evolutionary studies, and forensic science (2-5). The high copy number, and possibly the cellular location and molecular features of mtDNA, allow for increased recovery, thus providing a distinct advantage over nuclear DNA when working with highly compromised samples (6). The maternal inheritance and high mutation rate are characteristics extremely useful for evolutionary studies (7,8); in fact, mtDNA has been used to resolve evolutionary questions related to extinct species and to human migrations throughout the continents (9-12). The field of forensic science also relies upon mtDNA to identify missing persons, locate maternal relatives, identify victims in mass disasters, and, in some situations, include an individual at a crime scene (13-19).Early studies of the mtDNA genome revealed patterns of variation that were linked to geographic regions. Individuals with the same sequence variations were clustered into haplogroups defined by mutations at particular nucleotide positions (20-27). A closer examination of the mtDNA genomes of various populations led to the following assumptions: 1) several of the mtDNA mutations are highly correlated with the ethnic and geographic origin of the individual, 2) all mutations originated from a single mtDNA tree, and 3) the greatest variation and deepest root of the mtDNA tree is present in the African population. Furthermore, a calculation of the variation between mtDNA haplogroups demonstrated that 35% of the mutations were continent-specific, and therefore useful indicators of geographic origin (24,25,28-31).Before the advent of modern sequencing methods, the primary approach to identifying polymorphic sites throughout the mtDNA coding region was restriction fragment length polymorphism (RFLP) analysis. While this methodology is still utilized in certain contexts, direct sequencing of the mtDNA molecule is rapidly gaining acceptance as the method of choice for haplogroup typing (20,23,30,32-35). Single base primer extension, also known as minisequencing, is an example of a direct sequencing technique that is currently utilized for mtDNA haplogroup typing (36-42). This methodology, described in detail by Fiorentino et al (43), offers several advantages to the investigator over RFLP and conventional sequence analysis methods including the use of small amplicons (<150 bp), increased sensitivity and robustness, and multiplexing capability. Multiplexing capability is particularly important, especially in regard to forensic DNA analysis, as it reduces sample consumption while increasing throughput of sample processing and data analysis. Increased sensitivity allows for improved amplification success with DNA samples that contain limited starting template. Additionally, the possibility for high throughput processing can aid in population screening studies in situations where numerous samples need to be typed (44). This is particularly true in mass disaster or other mass screening situations, where a simple and rapid population screening tool that consumes little extract could effectively direct subsequent identification testing. In these situations, coding region sequencing would be expensive and time-consuming, and the subsequent data analysis a lengthy, burdensome, and potentially error-prone process (42,45-48). Furthermore, the possibility of obtaining interpretable results from poor quality polymerase chain reaction (PCR) products while simultaneously typing several polymorphisms throughout the mtDNA genome make it a more feasible method than conventional PCR fragment sequence analysis, especially in forensic cases and anthropological studies involving highly degraded or otherwise compromised human remains (16,36-38,49,50).In this article, we describe the development of a multiplex assay designed to simultaneously type twelve polymorphic positions located throughout the coding region of the mtDNA genome for the identification of haplogroups A, B, C, D, E, F, G, H, I, L1/L2, L3, M, N, and X. The intended utility of this assay is haplogroup typing of highly degraded human remains for either forensic casework or as a low cost, high throughput alternative for screening anthropological specimens. Validation of this assay included the analysis of 20 samples previously haplogroup typed by RFLP, and 94 samples for which haplogroup had been inferred based on control region (CR) sequence data. Additionally, and to evaluate the potential application of the assay for population screening, 31 samples were tested for which only the population of origin, and not the mtDNA haplogroup, was known. Finally, to verify robustness and sensitivity of the assay, we also tested two highly degraded human bones embalmed and buried in the early 1950s.     

Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection

Sindbis virus (SINV) is an arthropod-borne alphavirus found widely in Eurasia, Africa and Oceania. Clinical SINV infection, characterized by rash and arthritis, is reported primarily in Northern Europe. The laboratory diagnosis of SINV infection is based currently on serology. A one-step TaqMan^® real-time RT-PCR assay was developed for the detection of SINV and evaluated its clinical performance with acute-phase serum samples. The specificity and sensitivity of the real-time PCR assay were assessed using cell cultured Finnish SINV strains. The applicability of the assay for diagnostic use was evaluated using 58 serum samples from patients infected with SINV. The real-time RT-PCR assay was specific and sensitive for the detection of SINV in cell culture supernatants with a 95% detection limit of 9 genome copies/reaction determined by probit analysis. However, in the assay only 7/58 (12%) of serum samples were positive of which two were also positive by conventional nested PCR assay and none by virus isolation. This novel assay is specific and sensitive for detection of SINV and can be used for example for screening SINV in wildlife. However, molecular diagnostic techniques using serum samples seem to be of limited value for the diagnosis of human SINV infection due to the short and low viraemia of infection with SINV.