scholarly journals Epidemiological analysis of Porcine Viral Diarrhea Pathogens in Local Area

2020 ◽  
Author(s):  
Ying Shi ◽  
Benqiang Li ◽  
Jie Tao ◽  
Jinghua Cheng ◽  
Huili liu
Keyword(s):  
Prevention And Control ◽  
Detection Method ◽  
Multiple Infection ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Multiplex Detection ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Pcr Method ◽  
And Control

Abstract Background Porcine viral diarrhea can cause great damage to the pig industry and high mortality to piglets. Furthermore, multiple pathogen infections and synergistic infections commonly existed in clinic. This has resulted in great difficulties in determining the main pathogenic factors, which would delay the prevention and control of diseases. Methods A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017 and used for pathogen detection. A Luminex xTAG multiplex detection method was developed for the detection of 11 viral diarrhea pathogens, which allows for the simultaneous qualitative and quantitative detection of viral diarrhea pathogens in clinical samples. Results The minimum detection rate of the Luminex xTAG multiplex detection method was at least 10 times higher than the traditional PCR method. As a result, 209 (40.3%) were positive for porcine kobuvirus (PKoV), 138 (26.6%) for porcine astrovirus (PAstV), 91 (17.6%) for porcine epidemic diarrhea virus (PEDV), 78 (15.1%) for porcine sapelovirus (PSV), 68 (13.1%) for porcine sapovirus (PoSaV), 27 (5.2%) for porcine teschovirus (PTV), 25 (4.8%) for porcine deltacoronavirus (PDCoV), 17 (3.3%) for porcine rotavirus (PoRV), 16 (3.1%) for transmissible gastroenteritis virus (TGEV), 10 (1.9%) for porcine torovirus (PToV), and 9 (1.7%) for bovine viral diarrhea virus (BVDV), respectively. Furthermore, multiple infection rate of diarrhea sample was 17.57% for dual-infection, 11.58%for triple-infection, 4.63% for quadruple-infection, 0.77% for quintuple-infection, 0.58% for sextuple-infection and septuple-infection, respectively. Infection pattern of the viral diarrheal pathogens was changing, and different farm had the various diarrhea infection patterns, which proved the great importance of epidemiological surveillance and the guidance effect to clinical production. PoSaV, PoRV, PAstV, PToV and PEDV were indicated as the predominant viruses of clinical samples collected in 2017 by the quantitative analysis. Conclusions Here we provide a Luminex xTAG multiple detection method for viral diarrhea pathogen infection in clinical,which was more sensitive and specific than general multiplex PCR method. Furthermore, the surveillance confirmed the complicated infection status in China, which demonstrated the need for continuous surveillance and provided data for the prevention and control of viral diarrhea.

scholarly journals The Multiple Infections of Porcine Diarrhea Viruses in Local Area Based on A Luminex xTAG Multiplex Detection Method

2020 ◽  
Author(s):  
Huili liu ◽  
Ying Shi ◽  
Benqiang Li ◽  
Jie Tao ◽  
Jinghua Cheng
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Local Area ◽  
Clinical Samples ◽  
Multiple Infections ◽  
Quantitative Detection ◽  
Multiplex Detection ◽  
Viral Diarrhea ◽  
Infection Pattern ◽  
Pcr Method

Abstract Background Porcine viral diarrhea can cause great damage to the pig industry. However, multiple infections have contributed to the poor control of diarrhea, which has also resulted in great difficulties in determining the main pathogenic factors. Methods A Luminex xTAG multiplex detection method was developed for the detection of 11 viral diarrhea pathogens, which allows for the simultaneous qualitative and quantitative detection of viral diarrhea pathogens in clinical samples. A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017, and the pathogen spectrums and co-infections were analyzed. Results The minimum detection rate of the Luminex xTAG multiplex detection method was at least 10 times higher than the traditional PCR method. Of the 518 diarrhea samples, PEDV was found in 17.57% (91/518), PKoV in 40.35% (209/518), PAstV in 26.64% (138/518), PSV in 15.06% (78/518), PoSaV in 13.13% (68/518), PTV in 5.21% (27/518), PDCoV in 4.83% (25/518), PoRV in 3.28% (17/518), TGEV in 3.09% (16/518), PToV in 1.93% (10/518), and BVDV in 1.74% (9/518), respectively. Furthermore, multiple infections were commonly seen, with positive rate of 35.14%. Infection pattern of the viral diarrheal pathogens in a specific farm was changing, and different farms had the various diarrhea infection patterns. A longitudinal investigation showed that PEDV was still the key pathogen which was closely related to the death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease. Conclusions Here we provided a Luminex xTAG multiple detection method for viral diarrhea pathogen infection in clinical, which was more sensitive and specific than general multiplex PCR method. Furthermore, the surveillance confirmed high infection rate of PKoV, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status in southern China, suggesting that controlling porcine diarrhea might be more complex than previously thought. A better understanding of viruses that cause diarrhea in piglets will aid in better preventing and controlling epidemics of viral porcine diarrhea.

scholarly journals Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Jacqueline King ◽  
Anne Pohlmann ◽  
Kamila Dziadek ◽  
Martin Beer ◽  
Kerstin Wernike
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Economic Losses ◽  
Clinical Samples ◽  
Bovine Viral Diarrhea ◽  
Sequence Information ◽  
Whole Genome ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Loads

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

scholarly journals Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

2020 ◽  
Author(s):  
zhenhua Guo ◽  
Kunpeng Li ◽  
Songlin Qiao ◽  
Xinxin Chen ◽  
Ruiguang Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Limit Of Detection ◽  
African Swine Fever ◽  
Economic Losses ◽  
Clinical Samples ◽  
Diagnosis Method ◽  
Pcr Method ◽  
And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

scholarly journals Vaccination Failure in Eradication and Control Programs for Bovine Viral Diarrhea Infection

2021 ◽  
Vol 8 ◽  
Author(s):  
Aleksandra Antos ◽  
Pawel Miroslaw ◽  
Jerzy Rola ◽  
Miroslaw Pawel Polak
Keyword(s):  
Financial Burden ◽  
Bovine Viral Diarrhea ◽  
Serological Response ◽  
Prophylactic Measure ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Control Programs ◽  
Efficient Protection ◽  
Cattle Herds ◽  
And Control

Vaccination against bovine viral diarrhea (BVD) is one of the key elements to protect cattle herds from this economically important disorder. Bovine viral diarrhea virus (BVDV) is a pestivirus infecting animals at all ages with significant impact on reproductive, digestive, and respiratory systems. Financial burden caused by this pathogen prompts many farmers to introduce vaccination as the control and prophylactic measure especially when persistently infected (PI) individuals, being the main source of the virus in the herd, are removed after test-and-cull approach. The aim of the study was to compare the serological response in cattle herds where new PI calves were identified without prior removal of PI animals or despite their removal and after the introduction of whole herd vaccination against BVDV infection. Overall seroprevalence in 5 vaccinated herds was 91.7 and 83.3% using ELISA and virus neutralization test, respectively. Despite high titers for both vaccine and field strains of BVDV in analyzed herds the analysis of comparative strength of neutralization indicated that 41.4% of positive samples did not have a predominant titer against one specific subtype of BVDV. In 3 herds BVDV-1b subtype was identified while in 2 others it was BVDV-1d, while the vaccine used was based on BVDV-1a which was never identified in Poland so far. To increase the success of the BVDV eradication program, a careful approach is suggested when planning herd vaccination. Comparison of existing field strains and their similarity with vaccine strains at antigenic and genetic levels can be a useful approach to increase the effectiveness of vaccination and efficient protection of fetuses from persistent infection.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals Immunohistochemical Detection of Porcine Epidemic Diarrhea Virus Compared to Other Methods

1998 ◽  
Vol 5 (3) ◽  
pp. 412-414 ◽  
Author(s):  
Franco Guscetti ◽  
Curzio Bernasconi ◽  
Kurt Tobler ◽  
Kristien Van Reeth ◽  
Andreas Pospischil ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Good Reliability ◽  
Diarrhea Virus ◽  
Pcr Method ◽  
Porcine Epidemic Diarrhea ◽  
Cryostat Sections ◽  
Formalin Fixed

ABSTRACT An immunohistochemistry method using formalin-fixed tissues, a direct immunofluorescence method using cryostat sections, an enzyme-linked immunosorbent assay (ELISA), and a PCR method were compared for diagnosis in a litter of weaned pigs that had been experimentally inoculated with wild-type porcine epidemic diarrhea virus (PEDV) and killed between 6 and 60 h after onset of diarrhea. The immunohistochemistry method proved to be as reliable as direct immunofluorescence for diagnosis of PEDV in tissues collected postmortem. The good reliability of ELISA for investigating clinical samples was confirmed, whereas the PCR method used was ineffective.

Extraction of norovirus based on nanomagnetic beads and establishment of a fluorescence quantitative detection method

2020 ◽  
Vol 10 (9) ◽  
pp. 1463-1469
Author(s):  
Hui Chen ◽  
Kai Liu ◽  
Ziqi Xiao ◽  
Shaoyong Chen ◽  
Hang Liu ◽  
...  
Keyword(s):  
Acute Gastroenteritis ◽  
Detection Method ◽  
Melting Curve ◽  
Reaction System ◽  
Threshold Value ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Effective Prevention ◽  
Positive Stool ◽  
Pcr Method

Norovirus infection is the main cause of epidemic acute gastroenteritis outbreaks. As a result, norovirus merits careful attention. Acute gastroenteritis caused by norovirus is very harmful, necessitating effective prevention and monitoring. This study extracted norovirus using the Fe3O4 nanomagnetic bead method while establishing a rapid and sensitive quantitative norovirus detection method for emergency use. Norovirus RNA was extracted from norovirus-positive stool samples, norovirus-specific primers were designed, and SYBR Green I fluorescent dye was used to construct specific fluorescence quantitative reverse-transcription polymerase chain reaction (RT-PCR) method. Norovirus in the sample was successfully detected with the quantitative RT-PCR method established in this study, with a measured cycle threshold value of 28.73; the melting temperature value of the PCR product was approximately 83.5 °C. Additionally, by analyzing melting curve with quantitative PCR and evaluating the repeatability of the detection method, the performance of the reaction system in terms of specificity and repeatability was determined to be good. The fluorescence quantitative detection method established here can rapidly and effectively detect norovirus, and the method has good specificity and high repeatability. Currently, there is a trend of norovirus infection becoming increasingly severe. Thus, this rapid and sensitive fluorescence quantitative detection method will play a significant role in the monitoring and detection of norovirus and is thus worthy of promotion and application.

scholarly journals Rapid Detection of Aspergillus fumigatus Using Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Lateral Flow

2021 ◽  
Vol 11 ◽  
Author(s):  
Luxi Jiang ◽  
Xiaomeng Li ◽  
Rumeng Gu ◽  
Deguang Mu
Keyword(s):  
Aspergillus Fumigatus ◽  
Detection Method ◽  
Culture Method ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Specific Detection ◽  
Minimum Concentration ◽  
Whole Process ◽  
Multiple Cross ◽  
Pcr Method

Aspergillus fumigatus is an opportunistic, ubiquitous, saprophytic mold which can cause infection in the lungs, nose, eyes, brain, and bones in humans, especially in immunocompromised patients. However, it is difficult to diagnose A. fumigatus infection quickly. Here, we introduce a new detection method, namely multiple cross displacement amplification (MCDA) combined with nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB), which was proved to be fast, reliable, and simple for detecting A. fumigatus. We designed a set of 10 primers targeting the gene annexin ANXC4 of A. fumigatus. The best MCDA condition is 66 °C for 35 min. The minimum concentration that can be detected by this method was 10 fg. In the case of 100 sputum samples, 20 (20%) and 15 (15%) samples were positive by MCDA-LFB and PCR method, respectively. MCDA-LFB and traditional culture method showed the same results. Compared with the culture method, the diagnostic accuracy of MCDA-LFB can reach 100%. It showed that the MCDA-LFB method has better detection ability than the PCR method. We found that the whole process could be controlled within 60 min including the preparation of DNA (20 min), MCDA reaction (35 min) and results reporting (2 min). These results show that this assay is suitable for the rapid, sensitive and specific detection of A. fumigatus in clinical samples.

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