A novel and cost-effective real-time RT-PCR targeting 24 nucleotides deletion to differentiate PEDV wild-type and classical attenuated vaccine strains.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the swine industry, causing severe disease and resulting in substantial economic losses. Despite China's implementation of a large-scale vaccine immunization strategy in recent years, various strains of PEDV, including classical attenuated vaccine strains, continue to emerge in immunized pig herds. Here, we established a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains. This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, and we also explore the causes of this discriminatory target deficiency of this method through phylogenetic and recombination analysis. We found that these three classical attenuated vaccine strains exhibit closer phylogenetic relationships and higher sequence similarity with five cell-adapted strains. Recombination analysis revealed that although recombination is widespread in the PEDV genome, the 24-nucleotide deletion site remains stable without undergoing recombination and can be utilized as a target for identification. Further analysis revealed there are no enzyme cleavage sites near the 24-nucleotide site, suggesting that this deletion may have been lost during the process of culturing these viral strains in cells.The detection method we have established exhibits high specificity and sensitivity to PEDV, without cross-reactivity with other viruses causing diarrheal diseases. A total of 117 swine fecal samples were analyzed using this established one-step real-time reverse transcription PCR assay, indicating the presence of classical attenuated vaccine strains in pig herds in Gansu province, China. Additionally, the designed primer pairs and two probes can be placed in a single reaction tube to differentiate between these two types of strains, effectively reducing detection costs. These findings offer an efficient and cost-effective technological platform for clinical rapid identification testing of both wild-type and classical attenuated vaccine strains of PEDV, as well as for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.

Qualitative real-time RT-PCR assay for nOPV2 poliovirus detection.

Based on the success of the Sabin2-based vaccine, a next-generation nOPV2 poliovirus vaccine has been developed. For epidemic monitoring and conducting epidemiological investigations, it is necessary to have a diagnostic assay with the ability to differentiate this variant from others. Here we describe such a real-time RT-PCR assay. The region with the cre insertion in the 5'-UTR was chosen as the target, and the limit of detection was 103 copies/mL (2.5×103 copies/mL using Probit analysis) determined using armored RNA particles. Sensitivity and specificity were 86.28 - 100 % and 76.84 - 100 %, respectively (with 95 % CI). Thus, this method can be effectively used when it is necessary to make a differential diagnosis of poliovirus strains.

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

Evaluating an extraction-free sample preparation method for multiplex detection of SARS-Cov-2, influenza A/B, and RSV with implementation on a microfluidic chip.

Following the relaxation of COVID-19 restrictions, other respiratory viruses such as influenza and respiratory syncytial virus (RSV), whose transmission were decreased due to COVID-19 precautions, are rising again. Because of similar clinical features and reported co-infections, multiplex detection of SARS-CoV-2, influenza A/B, and RSV is required to use specific treatments. This study assessed an extraction-free sample preparation (heat treatment at 95°C for 3 minutes) for multiplex detection using rRT-PCR. Despite an observed Ct-delay (∆Ct) averageing 1.26 compared to the standard method, an acceptable total sensitivity of 92 % and a negative predictive value (NPV) of 96 % were obtained. Moreover, Implementation on a microfluidic chip demonstrated efficiency, maintaining an excellent correlation (R2=0.983) with the standard method. Combining this extraction-free procedure with rRT-PCR on a microfluidic chip seems promising, because it simplifies the design and reduces the cost and complexity of the integrated assay for multiplex detection of SARS-CoV-2, influenza A/B, and RSV.

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

TGFß signaling pathway in salivary gland tumors.

OBJECTIVE: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß cascade. DESIGN: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. RESULTS: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFß signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. CONCLUSIONS: Our study demonstrated the differential expression of TGFß cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection.

The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48-299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95-36.60) and 25.94 (range 16.37-36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman's ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

Accurate detection of influenza A virus by use of a novel cross-priming isothermal amplification-based point-of-care assay.

Influenza virus is known to cause respiratory tract infections of varying severity in individuals of all ages. The EasyNAT Rapid Flu assay is a newly developed in vitro diagnostic test that employs cross-priming isothermal amplification (CPA) to detect and differentiate influenza A and B viruses in human nasopharyngeal (NP) swabs. The aim of this study is to determine the performance characteristics of the EasyNAT Rapid Flu assay for rapid detection of influenza virus. The limit of detection (LOD) and cross-reactivity of the EasyNAT Rapid Flu assay were assessed. The clinical performance of the assay was evaluated using NP swab samples that were tested with real-time reverse-transcription polymerase chain reaction (RT-PCR) and Xpert Xpress Flu/RSV assay. The LOD for the detection of influenza A and B using the EasyNAT Rapid Flu assay was found to be 500 copies/mL. Furthermore, the assay exhibited no cross-reactivity with other common respiratory viruses tested. For the 114 NP swab samples tested for influenza A using both the EasyNAT Rapid Flu assay and real-time RT-PCR, the two assays demonstrated a high level of agreement (κ = 0.963, P < 0.001), with a positive percentage agreement (PPA) of 97.7% and a negative percentage agreement (NPA) of 98.6%. Similarly, for the 43 NP swab samples tested for influenza A and B using both the EasyNAT Rapid Flu assay and Xpert Xpress Flu/RSV assay, the two assays showed a high level of agreement (κ = 0.933, P < 0.001), with the overall rate of agreement (ORA) of 97.7% for influenza A and 100% for influenza B. The EasyNAT Rapid Flu assay demonstrates excellent performance in the detection of influenza A, highlighted by its strong agreement with RT-PCR-based assays.IMPORTANCEThe newly developed EasyNAT Rapid Flu assay is an innovative cross-priming isothermal amplification-based method designed for detecting influenza A and B viruses at point-of-care settings. This study aims to thoroughly assess the analytical and clinical performance of the assay, offering valuable insights into its potential advantages and limitations. The findings of this research hold significant implications for clinical practice.

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses.

IMPORTANCE: Since the pandemic of coronavirus diseases 2019, the use of real-time PCR assay has become widespread among people who were not familiar with it in virus detection. As a result, whether a high real-time PCR value in one time test indicates virus transmissibly became a complicated social problem, regardless of the difference in assays and/or amplification conditions, the time and number of diagnostic test during the time course of infection. In addition, the multiple positives in the test of respiratory viruses further add to the confusion in the interpretation of the infection. To address this issue, we performed virus isolation using pediatric SARI (severe acute respiratory infections) specimens on air-liquid interface culture of human bronchial/tracheal epithelial cell culture. The result of this study can be a strong evidence that the specimens showing positivity for multiple agents in real-time PCR tests possibly contain infectious viruses.

Dissociation and Culture of Adult Mouse Satellite Glial Cells.

Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells. They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system. Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection. In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain. Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently. This has impeded further studies of their function and the testing of new therapeutics. This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity. We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging. Key features • Detailed and simplified protocol to dissociate and culture primary satellite glial cells (SGCs) from adult mice. • Cells are dissociated in approximately 2-3 h and cultured for approximately two weeks. • These SGC cultures allow both molecular and functional studies.

Establishing a molecular toolbox of lineage-specific real-time RT-PCR assays for the characterization of foot-and-mouth disease viruses circulating in Asia.

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

Optimization of RT-PCR methods for enterovirus detection in groundwater.

Enteroviruses (EVs), which belong to the Picornaviridae family, infect individuals asymptomatically or cause mild symptoms (fever, runny nose, cough, skin rash, sneezing, mouth blister). Severe cases can cause various diseases, such as acute hemorrhagic conjunctivitis, aseptic meningitis, or myocarditis, especially in infants. These viruses can be transmitted via the fecal-oral route via contaminated water. In this study, we established a polymerase chain reaction (PCR) method for detecting EVs in water sample using Coxsackievirus B5 (CV-B5) and Echovirus 30 (E-30), which belong to species B of the four species of EVs (EV-A to D). Several methods have been investigated and compared for the detection of EVs, including real-time reverse transcription (RT) polymerase chain reaction and conventional RT-PCR. The most sensitive primer sets were selected, and the PCR conditions were modified to increase sensitivity. We also quantified the detection limits of real-time and conventional RT-PCR. The detection limits of conventional RT-PCR were detected in 105-106 copy/mL for CV-B5 and 106-107 copy/mL for E-30, respectively. This optimized method for detecting EVs is expected to contribute substantially to the investigation of EV outbreaks in water samples.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays.

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Development of a Novel Korean H9-Specific rRT-PCR Assay and Its Application for Avian Influenza Virus Surveillance in Korea.

Since the 2000s, the Y439 lineage of H9N2 avian influenza virus (AIV) has been the predominant strain circulating in poultry in Korea; however, in 2020, the Y280 lineage emerged and spread rapidly nationwide, causing large economic losses. To prevent further spread and circulation of such viruses, rapid detection and diagnosis through active surveillance programs are crucial. Here, we developed a novel H9 rRT-PCR assay that can detect a broad range of H9Nx viruses in situations in which multiple lineages of H9 AIVs are co-circulating. We then evaluated its efficacy using a large number of clinical samples. The assay, named the Uni Kor-H9 assay, showed high sensitivity for Y280 lineage viruses, as well as for the Y439 lineage originating in Korean poultry and wild birds. In addition, the assay showed no cross-reactivity with other subtypes of AIV or other avian pathogens. Furthermore, the Uni Kor-H9 assay was more sensitive, and had higher detection rates, than reference H9 rRT-PCR methods when tested against a panel of domestically isolated H9 AIVs. In conclusion, the novel Uni Kor-H9 assay enables more rapid and efficient diagnosis than the "traditional" method of virus isolation followed by subtyping RT-PCR. Application of the new H9 rRT-PCR assay to AI active surveillance programs will help to control and manage Korean H9 AIVs more efficiently.

Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification.

Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing - such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range. This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (SLC4A1) and menstrual fluid (STC1), saliva (HTN3) and vaginal material (CYP2B7P), and spermatozoa (PRM1) and seminal fluid (KLK2), all including a synthetic internal control RNA. Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R2 > 0.99), and high precision (most mean CV < 1 %). STC1 showed some instances of sporadic expression in blood, semen, and vaginal material at high CT values. CYP2B7P showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01-0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab. A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target CYP2B7P expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.

Monomeric myeloperoxidase is a specific biomarker for early-stage ovarian cancer.

Background: Ovarian cancer cells are known to express myeloperoxidase (MPO), an oxidant-producing enzyme with a 150 kDa homodimer, consisting of two identical monomers connected by a disulfide bond. Here, we aim to validate monomeric MPO (mMPO) as a biomarker for the early detection of ovarian cancer.Methods: Human ovarian cancer cells, sera from patients at various stages, sera from non-cancer inflammatory gynecological diseases, and healthy volunteers were used. Monomeric and dimeric MPO were measured by ELISA. Receiver operating curves were used to compare the predictive powers of serum dimeric and monomeric MPO to discriminate between samples.Results: The expression of MPO was unique to ovarian cancer cells. Specifically, mMPO was found to be the only form of MPO in all ovarian cancer cell lines. Intriguingly, mMPO was detected in the sera from all patients with ovarian cancer at various stages, but not from healthy individuals. Serum mMPO discriminated between early-stage ovarian cancer, healthy controls, and benign inflammatory gynecologic disorders. In addition, mMPO discriminated between the early and late stages of the disease.Conclusion: This work highlights mMPO as a potential biomarker for early detection of ovarian cancer, which is critically needed.