A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16.

BACKGROUND: Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. METHODS: Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. RESULTS: The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. CONCLUSIONS: The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

BACKGROUND: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control. METHODS: We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min. RESULTS: The analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol. CONCLUSIONS: We provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.

A case control study on the prevalence of enterovirus in children samples and its association with diarrhea.

Some serotypes of enterovirus (EV) may lead to transient and symptomatic gastrointestinal infections while others are commensal residents of the human gut. To determine whether certain EV types are more often associated with diarrhea, we conducted a preliminary study on the prevalence of EV serotypes and common diarrhea viruses in fecal samples of diarrhea children and healthy controls. EV was tested with one step nest polymerase chain reaction and typed by direct sequencing while common causative diarrhea viruses rotavirus (RV), norovirus (NoV), adenovirus (AdV), bocavirus (HBoV), and astrovirus (AstV) were screened with multiplex PCR assays. Human Rhinovirus (HRV) and human EVs that were present in both groups were further quantified and their odds ratios (OR) were calculated. Enteric pathogens were detected in 89 (32.6%) of 273 children with diarrhea and included human EVs (51, 18.68%), HRV (32, 11.72%), RV (38, 13.92%), AdV (24, 8.79%), NoVGII (16, 8.79%), HBoV (8, 2.93%) and AstV (3, 1.09%). Potential enteric pathogens were found in 25 (6.93%) of 361 healthy controls and included human EV (59, 16.34%), HRV (8, 2.22%), RV (1, 0.28%), NoVGII (5, 1.39%), AstV (2, 0.55%), AdV (16, 4.43%) and HBoV (1, 0.28%). In addition, EV71, echovirus 3,9,14,25 and coxsackievirus A14 existed in healthy controls only, while HRV, echovirus11,18, coxsackievirus A2,4,6 and B2,4 were found in both patients and healthy controls. OR assessment confirmed a strong association of HRV (P < 0.001) and a weak one for echovirus 11 and coxsackievirus A6 with diarrhea (P > 0.05). Our results indicate the diversity of EV serotypes in diarrhea and healthy control groups varies, and the potential etiological role of HRV in diarrhea.

A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction.

BACKGROUND: Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. METHODS: Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). RESULTS: A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. CONCLUSIONS: We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.

A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

BACKGROUND: Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. METHODS: In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. RESULTS: The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). CONCLUSION: The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

Adenovirus associated with acute diarrhea: a case-control study.

BACKGROUND: Diarrhea is a major source of morbidity and mortality among young children in low-income and middle-income countries. Human adenoviruses (HAdV), particular HAdV species F (40, 41) has been recognized as important causal pathogens, however limited data exist on molecular epidemiology of other HAdV associated with acute gastroenteritis. METHODS: In the present preliminary study, we performed a case-control study involving 273 children who presented diarrheal disease and 361 healthy children matched control in Children's hospital of Hebei Province (China) to investigate the relationship between non-enteric HAdV and diarrhea. HAdV were detected and quantified using quantitative real-time PCR (qPCR) and serotyped by sequencing and phylogenetic analysis. Odds ratio (OR) was used to assess the risk factor of HAdV. RESULTS: HAdV were detected in 79 (28.94%) of 273 children with diarrhea including 7 different serotypes (HAdV 40, 41, 3, 2,1,5 and 57) with serotypes 40, 41 and 3 being the most dominant and in 26 (7.20%) of 361 healthy children containing 9 serotypes (HAdV 40, 41, 3, 2,1,5,57,6 and 31). A majority (91.14%) of HAdV positives occurred in diarrhea children and 65.38% in controls< 3 years of age. No significant difference in the viral load was found between case and control groups or between Ad41-positive patients and healthy controls. In addition to HAdV 40 and 41, HAdV 3 was also associated with diarrhea (OR = 17.301, adjusted OR = 9.205, p < 0.001). CONCLUSIONS: Our results demonstrate a high diversity of HAdV present among diarrhea and healthy children and implicate that non-enteric HAdV3 may lead to diarrhea.

VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens.

OBJECTIVE: Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. METHODS: A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. RESULTS: A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. CONCLUSION: The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer.

OBJECTIVE: Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. METHODS: In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. RESULTS: Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. CONCLUSION: MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Molecular epidemiology of reemergent rabies in Yunnan Province, southwestern China.

Yunnan Province in China borders 3 countries (Vietnam, Laos, and Myanmar) in Southeast Asia. In the 1980s, a large-scale rabies epidemic occurred in this province, which subsided by the late 1990s. However, 3 human cases of rabies in 2000 indicated reemergence of the disease in 1 county. In 2012, rabies was detected in 77 counties; 663 persons died of rabies during this new epidemic. Fifty two rabies virus strains obtained during 2008-2012 were identified and analyzed phylogenetically by sequencing the nucleoprotein gene. Of the 4 clades identified, clades YN-A and YN-C were closely related to strains from neighboring provinces, and clade YN-B was closely related to strains from Southeast Asia, but formed a distinct branch. Rabies virus diversity might be attributed to dog movements among counties, provinces, and neighboring countries. These findings suggest that Yunnan Province is a focal point for spread of rabies between Southeast Asia and China.

Human rabies surveillance and control in China, 2005-2012.

BACKGROUND: Rabies reemerged in China during the 1990s with a gradual increase in the number and geographical dispersion of cases. As a consequence, a national surveillance program was introduced in 2005 to investigate the outbreak in terms of vaccination coverage, PEP treatment, and geographical and social composition. METHODS: The surveillance program was coordinated at the national level by the Chinese Center for Disease Control (CCDC) with data collected by regional health centres and provincial CCDCs, and from other official sources. Various statistical and multivariate analysis techniques were then used to evaluate the role and significance of implemented policies and strategies related to rabies prevention and control over this period. RESULTS: From 2005-2012, 19,221 cases were reported across 30 provinces, but these primarily occurred in rural areas of southern and eastern China, and were predominantly associated with farmers, students and preschool children. In particular, detailed analysis of fatalities reported from 2010 to 2011 shows they were associated with very low rates of post exposure treatment compared to the cases with standard PEP. Nevertheless, regulation of post-exposure prophylaxis quality, together with improved management and vaccination of domesticated animals, has improved prevention and control of rabies. CONCLUSIONS: The various control policies implemented by the government has played a key role in reducing rabies incidences in China. However, level of PEP treatment varies according to sex, age, degree and site of exposure, as well as the source of infection. Regulation of PEP quality together with improved management and vaccination of domesticated animals have also helped to improve prevention and control of rabies.

Factors influencing the number of rabies cases in children in China.

To understand the epidemic situation and factors influencing rabies cases in children in China, we obtained an overview of the current epidemic based on individual data of rabies cases in children and a descriptive analysis was carried on the prevalence and related factors. The results showed that the rabies cases in children accounted for 21.3% of the total number of rabies cases in China, 97.0% of these cases occurred in rural areas, they were mainly caused by dogs (81.5%), and were primarily level III exposure (47.7%). More than half of the cases were not treated with wound care, vaccination rate was extremely low (15.7%), and only 5.9% of cases were injected with antibodies. Furthermore, 25.4% of cases adopted incorrect treatments such as extruding bleed and wound closure, cases vaccinated with 5 injections accounted for only 22.5%. In conclusion, the prevalence of rabies cases in children in China remains a serious concern, the number and immune status of dogs in rural areas, and knowledge of rabies by risk populations should be considered in future rabies prevention and control programs.

Comparative analysis of the pathogenic mechanisms of street rabies virus strains with different virulence levels.

OBJECTIVE: To characterize two strains of street rabies virus (RABV) isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determined to reveal variation in the pathogenesis of infection between street rabies virus strains. METHODS: Ten-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups. RESULTS: Infection with the street virus strain CNM1101C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain, CNM1103C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused severe T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact. CONCLUSION: The two street rabies virus strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.

Identification of animal rabies in Inner Mongolia and analysis of the etiologic characteristics.

OBJECTIVE: To perform pathological observation and etiological identification of specimens collected from dairy cows, beef cattle and dogs which were suspected of rabies in Inner Mongolia in 2011, and analyze their etiological characteristics. METHODS: Pathological observation was conducted on the brain specimens of three infected animals with Hematoxylin-Eosin staining, followed by confirmation using immunofluorescence and nested RT-PCR methods. Finally, phylogenetic analysis was conducted using the virus N gene sequence amplified from three specimens. RESULTS: Eosinophilic and cytoplasmic inclusion bodies were seen in neuronal cells of the CNS; and rabies non-characteristic histopathological changes were also detected in the CNS. The three brain specimens were detected positive. N gene nucleotide sequence of these three isolates showed distinct sequence identity, therefore they fell into different groups in the phylogenetic analysis. N gene in the cow and dog had higher homology with that in Hebei isolate, but that in the beef cattle had higher homology with that in Mongolian lupine isolate and Russian red fox isolate. CONCLUSION: Rabies were observed in the dairy cow, beef cattle and canine in the farm in Inner Mongolia, in 2011, which led to a different etiologic characteristics of the epidemic situation.

Risk Factors of Depression Screened by Two-Sample Mendelian Randomization Analysis: A Systematic Review.

Objective: This study explored the potentially modifiable factors for depression and major depressive disorder (MDD) from the MR-Base database and further evaluated the associations between drug targets with MDD. Methods: We analyzed two-sample of Mendelian randomization (2SMR) using genetic variant depression ( n = 113,154) and MDD ( n = 208,811) from Genome-Wide Association Studies (GWAS). Separate calculations were performed with modifiable risk factors from MR-Base for 1,001 genomes. The MR analysis was performed by screening drug targets with MDD in the DrugBank database to explore the therapeutic targets for MDD. Inverse variance weighted (IVW), fixed-effect inverse variance weighted (FE-IVW), MR-Egger, weighted median, and weighted mode were used for complementary calculation. Results: The potential causal relationship between modifiable risk factors and depression contained 459 results for depression and 424 for MDD. Also, the associations between drug targets and MDD showed that SLC6A4, GRIN2A, GRIN2C, SCN10A, and IL1B expression are associated with an increased risk of depression. In contrast, ADRB1, CHRNA3, HTR3A, GSTP1, and GABRG2 genes are candidate protective factors against depression. Conclusion: This study identified the risk factors causally associated with depression and MDD, and estimated 10 drug targets with significant impact on MDD, providing essential information for formulating strategies to prevent and treat depression.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology.

Objective: Recombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. Methods: Recombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. Results: The M-RAP method had sensitivity rates of 1, 10, and 1 copies/µL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively ( P < 0.05). Conclusion: An M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Multicenter evaluation of a simple and sensitive nucleic acid self-testing for SARS-CoV-2.

A rapid and accurate COVID-19 diagnosis is a prerequisite for blocking the source of infection as soon as possible and taking the appropriate medical action. Herein, we developed GeneClick, a device for nucleic acid self-testing of SARS-CoV-2, consisting of three modules: a sampling kit, a microfluidic chip-based disposable cartridge, and an amplification reader. In addition, we evaluated the clinical performance of GeneClick using 2162 nasal swabs collected at three medical institutions, using three commercial RT-qPCR kits and an antigen self-test as references. Compared to RT-qPCR, the sensitivity and specificity of the GeneClick assay were 97.93% and 99.72%, respectively, with a kappa value of 0.979 (P â€‹< â€‹0.01). Of the 2162 samples, 2076 were also tested for SARS-CoV-2 antigens. Among the 314 positive samples identified by GeneClick assay, 63 samples were undetected by antigen tests. Overall, the GeneClick nucleic acid self-test demonstrated higher accuracy than the antigen-based detection. Based on the additional features, including simple operation, affordable price, portable device, and reliability of smartphone APP-driven sampling and result reporting, GeneClick offers a powerful tool for field-based SARS-CoV-2 detection in primary healthcare institutions or at-home use.

Preparation and initial application of a monoclonal antibody specific for a newly discovered conserved linear epitope of rabies virus nucleoprotein.

OBJECTIVE: To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. METHODS: Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT). RESULTS: Two positive hybridoma cell lines, RVNP-mAb1-CL and RVNP-mAb2-CL, were obtained. RVNP- mAb1-CL produced a higher concentration of monoclonal antibody RVNP-mAb1 in Balb/c ascites. FITC-labeled RVNP-mAb1 showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR). CONCLUSION: FITC-labeled RVNP-mAb1 has potential application for laboratory diagnosis of rabies.

Development and evaluation of a sensitive recombinase aided amplification assay for rapid detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is a widely distributed pathogen in the coastal areas, which causes food poisoning and leads to gastroenteritis and sepsis. Therefore, developing a simple, sensitive, and rapid detection method for V. parahaemolyticus is a major concern globally. This study established a sensitive and rapid technique based on recombinase aided amplification (RAA) to detect V. parahaemolyticus. The RAA reaction was carried out successfully at 39 °C within 30 min. The sensitivity of the RAA assay was 101 copies/µL using the recombinant plasmid and 10-3 ng/µL using the V. parahaemolyticus strain. In addition, RAA directly detected 7 × 103 CFU/mL of simulated fecal samples and 0.1 CFU/mL after enrichment for 4 h. The sensitivity and specificity of the RAA assay using fecal and fish samples were 100% similar to that of the real-time PCR. We conclude that the RAA assay is an ideal screening method for detecting V. parahaemolyticus due to its rapidity, high accuracy, and simplicity in operation.