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Article ; Online: Universal primer multiplex PCR assay for detection and genotyping of porcine astroviruses.

Zhang, Qiuya / Wen, Dan / Liu, Qin / Opriessnig, Tanja / Yu, Xiaoya / Jiang, Yonghou

2023 Volume 322, Page(s) 114822

Abstract: Porcine astroviruses (PAstV) are members of the family Astroviridae, Mamastravirus genus and have been identified to have five genotypes (PAstV1-5). These viruses are highly prevalent in pigs and can cause enteric disease as well as neurological or ... ...

Abstract	Porcine astroviruses (PAstV) are members of the family Astroviridae, Mamastravirus genus and have been identified to have five genotypes (PAstV1-5). These viruses are highly prevalent in pigs and can cause enteric disease as well as neurological or respiratory symptoms depending on their genotypes. At present, the epidemiological impacts of some PAstV genotypes on pigs are largely unknown and hence continuously monitoring of these PAstVs may be needed. The purpose of this research was to develop an improved and efficient detection tool for PAstVs and to evaluate the developed method using clinical samples. Initially, a set of five chimeric primers (CP), each comprising genotype specific primer pairs with an identical universal adapter at the 5' end, and a universal primer (UP) that is identical to universal adapter sequence, were designed. With these tools in place, a novel multiplex PCR system with universal primer was established for the simultaneous detection of the five types of PAstV. This method can specifically detect PAstV genotypes, with a limit of detection (LOD) of 5 copies/μL for each genotype irrespective of single or mixed target template. Using this new assay, 273 pig fecal samples were investigated for further assay evaluation. Among all samples, the positive rate was 70.0% with PAstV4 in 56.8% of the samples, PAstV2 in 38.8%, PAstV1 in 16.8%, and PAstV5 in 11.0%. More than one PAstV in a sample were detected in 39.2% of the samples. The consistency rate between the novel multiplex PCR and singleplex PCRs was 96.4-100%. Given its rapidity, specificity and sensitivity, the novel multiplex PCR is a useful approach for demonstrating single or mixed genotype infections of PAstV.
MeSH term(s)	Animals ; Swine ; Astroviridae Infections ; Multiplex Polymerase Chain Reaction ; Genotype ; Swine Diseases/diagnosis ; Sensitivity and Specificity
Language	English
Publishing date	2023-09-18
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2023.114822
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Article ; Online: Multiplex gel-based PCR assay for the simultaneous detection of 5 genotypes of porcine astroviruses

Zhang, Qiuya / Liu, Qin / Opriessnig, Tanja / Wen, Dan / Gu, Keda / Jiang, Yonghou

Journal of Veterinary Diagnostic Investigation. 2023 Mar., v. 35, no. 2 p.132-138

2023

Abstract: Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1–5. To obtain information ... ...

Abstract	Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1–5. To obtain information on the epidemiology of PAstV, we established a multiplex PAstV PCR assay to detect and differentiate the 5 PAstV genotypes simultaneously. The assay utilized specific primers for each genotype, producing fragments of 307, 353, 205, 253, and 467 bp, representing PAstV1–5, respectively. Our multiplex PCR assay amplified all 5 DNA fragments from single or mixed viral genomes without cross-reactions with other PAstV genotypes or other viruses in pigs. The limit of detection of the multiplex PCR assay was 5 × 10² copies/μL for PAstV1 and PAstV4, and 5 × 10³ copies/μL for PAstV2, PAstV3, and PAstV5. We examined 76 pig fecal specimens with our multiplex PCR assay. PAstV was detected in 36 of 76 (47.4%) samples; ≥2 PAstVs were found in 20 of 76 (26.3%) samples. The multiplex PCR assay results were essentially the same as the results using a monoplex PAstV PCR assay, with a coincidence rate of >96%. Our multiplex PCR method provides a simple, sensitive, and specific detection tool for PAstV detection and epidemiologic surveys.
Keywords	DNA ; Mamastrovirus 3 ; detection limit ; diarrhea ; epidemiology ; genetic variation ; genotype ; polymerase chain reaction ; swine ; viral genome ; viruses ; detection ; multiplex PCR ; porcine astrovirus
Language	English
Dates of publication	2023-03
Size	p. 132-138.
Publishing place	SAGE Publications
Document type	Article ; Online
ZDB-ID	287603-6
ISSN	1943-4936 ; 1040-6387
ISSN (online)	1943-4936
ISSN	1040-6387
DOI	10.1177/10406387221145329
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Multiplex gel-based PCR assay for the simultaneous detection of 5 genotypes of porcine astroviruses.

Zhang, Qiuya / Liu, Qin / Opriessnig, Tanja / Wen, Dan / Gu, Keda / Jiang, Yonghou

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

2022 Volume 35, Issue 2, Page(s) 132–138

Abstract	Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1-5. To obtain information on the epidemiology of PAstV, we established a multiplex PAstV PCR assay to detect and differentiate the 5 PAstV genotypes simultaneously. The assay utilized specific primers for each genotype, producing fragments of 307, 353, 205, 253, and 467 bp, representing PAstV1-5, respectively. Our multiplex PCR assay amplified all 5 DNA fragments from single or mixed viral genomes without cross-reactions with other PAstV genotypes or other viruses in pigs. The limit of detection of the multiplex PCR assay was 5 × 10
MeSH term(s)	Animals ; Swine ; Astroviridae Infections/diagnosis ; Astroviridae Infections/epidemiology ; Astroviridae Infections/veterinary ; Multiplex Polymerase Chain Reaction/veterinary ; Swine Diseases/diagnosis ; Swine Diseases/epidemiology ; Genotype ; Sensitivity and Specificity
Language	English
Publishing date	2022-12-27
Publishing country	United States
Document type	Journal Article
ZDB-ID	287603-6
ISSN	1943-4936 ; 1040-6387
ISSN (online)	1943-4936
ISSN	1040-6387
DOI	10.1177/10406387221145329
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Article ; Online: Novel universal primer-pentaplex PCR assay based on chimeric primers for simultaneous detection of five common pig viruses associated with diarrhea.

Nan, Pei / Wen, Dan / Opriessnig, Tanja / Zhang, Qiuya / Yu, Xiaoya / Jiang, Yonghou

Molecular and cellular probes

2021 Volume 58, Page(s) 101747

Abstract: Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal ...

Abstract	Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5' end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification. Five universal primers with CPs (UP1-5) were designed and evaluated in an UP-based single PCR (UP-S-PCR). All five UPs were found to work efficiently and UP2 exhibited the best performance. After system optimizations, the analytical sensitivity of the UP-M-PCR, using plasmids containing the specific viral target fragments, was 5 copies/reaction for each of the five viruses irrespective of presence of a single or multiple viruses in the reaction. No cross-reaction was observed with other non-target viruses. When 273 fecal samples from clinically healthy pigs were tested, the assay sensitivity was 90.9-100%, the specificity was 98.0-100%, and the agreement rate with the UP-S-PCR was 98.5-99.6% with a Kappa value being 0.95-0.98. In summary, the UP-M-PCR developed here is a rapid and highly sensitive and specific detection method that can be used to demonstrate mixed infections in pigs with diarrhea.
MeSH term(s)	Animals ; Diarrhea/veterinary ; Polymerase Chain Reaction ; Porcine epidemic diarrhea virus/genetics ; Sensitivity and Specificity ; Swine ; Swine Diseases/diagnosis ; Viruses
Language	English
Publishing date	2021-06-08
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	639082-1
ISSN	1096-1194 ; 0890-8508
ISSN (online)	1096-1194
ISSN	0890-8508
DOI	10.1016/j.mcp.2021.101747
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Article: Novel universal primer-pentaplex PCR assay based on chimeric primers for simultaneous detection of five common pig viruses associated with diarrhea

Nan, Pei / Wen, Dan / Opriessnig, Tanja / Zhang, Qiuya / Yu, Xiaoya / Jiang, Yonghou

Elsevier Ltd Molecular and cellular probes. 2021 Aug., v. 58

2021

Abstract	Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5′ end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification. Five universal primers with CPs (UP1-5) were designed and evaluated in an UP-based single PCR (UP–S-PCR). All five UPs were found to work efficiently and UP2 exhibited the best performance. After system optimizations, the analytical sensitivity of the UP-M-PCR, using plasmids containing the specific viral target fragments, was 5 copies/reaction for each of the five viruses irrespective of presence of a single or multiple viruses in the reaction. No cross-reaction was observed with other non-target viruses. When 273 fecal samples from clinically healthy pigs were tested, the assay sensitivity was 90.9–100%, the specificity was 98.0–100%, and the agreement rate with the UP-S-PCR was 98.5–99.6% with a Kappa value being 0.95–0.98. In summary, the UP-M-PCR developed here is a rapid and highly sensitive and specific detection method that can be used to demonstrate mixed infections in pigs with diarrhea.
Keywords	Porcine circovirus-2 ; Porcine epidemic diarrhea virus ; Rotavirus A ; Transmissible gastroenteritis virus ; cross reaction ; detection limit ; diarrhea ; plasmids ; swine ; tail ; viruses
Language	English
Dates of publication	2021-08
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	639082-1
ISSN	1096-1194 ; 0890-8508
ISSN (online)	1096-1194
ISSN	0890-8508
DOI	10.1016/j.mcp.2021.101747
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis.

Wen, Dan / Liu, Gaopeng / Opriessnig, Tanja / Yang, Zongqi / Jiang, Yonghou

Journal of virological methods

2019 Volume 268, Page(s) 1–8

Abstract: In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous ... ...

Abstract	In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.
MeSH term(s)	Animals ; DNA Primers/genetics ; Diarrhea/veterinary ; Diarrhea/virology ; Feces/virology ; Gastrointestinal Tract/virology ; Multiplex Polymerase Chain Reaction ; Reproducibility of Results ; Sensitivity and Specificity ; Swine ; Swine Diseases/diagnosis ; Swine Diseases/virology ; Transition Temperature ; Virus Diseases/diagnosis ; Virus Diseases/veterinary ; Viruses/classification ; Viruses/isolation & purification
Chemical Substances	DNA Primers
Language	English
Publishing date	2019-03-04
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2019.03.001
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Spike-specific T cells are enriched in breastmilk following SARS-CoV-2 mRNA vaccination.

Armistead, Blair / Jiang, Yonghou / Carlson, Marc / Ford, Emily S / Jani, Saumya / Houck, John / Wu, Xia / Jing, Lichen / Pecor, Tiffany / Kachikis, Alisa / Yeung, Winnie / Nguyen, Tina / Coig, Rene / Minkah, Nana / Larsen, Sasha E / Coler, Rhea N / Koelle, David M / Harrington, Whitney E

Mucosal immunology

2023 Volume 16, Issue 1, Page(s) 39–49

Abstract: Human breastmilk is rich in T cells; however, their specificity and function are largely unknown. We compared the phenotype, diversity, and antigen specificity of T cells in breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 ...

Abstract	Human breastmilk is rich in T cells; however, their specificity and function are largely unknown. We compared the phenotype, diversity, and antigen specificity of T cells in breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 messenger RNA (mRNA) vaccination. Relative to blood, breastmilk contained higher frequencies of T effector and central memory populations that expressed mucosal-homing markers. T cell receptor sequence overlap was limited between blood and breastmilk. Overabundant breastmilk clones were observed in all individuals, were diverse, and contained complementarity-determining regions in three sequences with known epitope specificity, including to SARS-CoV-2 spike. SARS-CoV-2 spike-specific T cell receptors were more frequent in breastmilk compared to blood and expanded in breastmilk following a 3
MeSH term(s)	Infant ; Female ; Humans ; Milk, Human ; SARS-CoV-2 ; T-Lymphocytes ; Lactation ; COVID-19 ; Vaccination ; RNA, Messenger ; Antibodies, Viral
Chemical Substances	RNA, Messenger ; Antibodies, Viral
Language	English
Publishing date	2023-01-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2411370-0
ISSN	1935-3456 ; 1933-0219
ISSN (online)	1935-3456
ISSN	1933-0219
DOI	10.1016/j.mucimm.2023.01.003
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Article ; Online: Application of electrospun fibers for female reproductive health.

Blakney, Anna K / Jiang, Yonghou / Woodrow, Kim A

Drug delivery and translational research

2017 Volume 7, Issue 6, Page(s) 796–804

Abstract: Here, we present the current challenges in women's reproductive health and the current state-of-the-art treatment and prevention options for STI prevention, contraception, and treatment of infections. We discuss how the versatile platform of electrospun ... ...

Abstract	Here, we present the current challenges in women's reproductive health and the current state-of-the-art treatment and prevention options for STI prevention, contraception, and treatment of infections. We discuss how the versatile platform of electrospun fibers can be applied to each challenge, and postulate at how these technologies could be improved. The void of approved electrospun fiber-based products yields the potential to apply this useful technology to a number of medical applications, many of which are relevant to women's reproductive health. Given the ability to tune drug delivery characteristics and three-dimensional geometry, there are many opportunities to pursue new product designs and routes of administration for electrospun fibers. For each application, we provide an overview of the versatility of electrospun fibers as a novel dosage form and summarize their advantages in clinical applications. We also provide a perspective on why electrospun fibers are well-suited for a variety of applications within women's reproductive health and identify areas that could greatly benefit from innovations with electrospun fiber-based approaches.
Language	English
Publishing date	2017-12
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2590155-2
ISSN	2190-3948 ; 2190-393X
ISSN (online)	2190-3948
ISSN	2190-393X
DOI	10.1007/s13346-017-0386-3
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Article ; Online: Corrigendum to "Simultaneous measurement of etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC-MS/MS assay" [J. Chromatogr. B 1025 (2016) 110-118].

Blakney, Anna K / Jiang, Yonghou / Whittington, Dale / Woodrow, Kim A

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

2016 Volume 1039, Page(s) 88

Language	English
Publishing date	2016-11-09
Publishing country	Netherlands
Document type	Journal Article ; Published Erratum
ZDB-ID	1180823-8
ISSN	1873-376X ; 0378-4347 ; 1570-0232 ; 1387-2273
ISSN (online)	1873-376X
ISSN	0378-4347 ; 1570-0232 ; 1387-2273
DOI	10.1016/j.jchromb.2016.10.022
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Article: Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis

Wen, Dan / Liu, Gaopeng / Opriessnig, Tanja / Yang, Zongqi / Jiang, Yonghou

Journal of virological methods. 2019 June, v. 268

2019

Abstract	In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.
Keywords	Porcine circovirus-2 ; Porcine epidemic diarrhea virus ; Rotavirus ; Transmissible gastroenteritis virus ; detection limit ; diagnostic techniques ; diarrhea ; digestive system diseases ; farms ; financial economics ; fluorescent dyes ; melting ; melting curve analysis ; melting point ; mixed infection ; monitoring ; quantitative polymerase chain reaction ; swine ; swine diseases ; viruses
Language	English
Dates of publication	2019-06
Size	p. 1-8.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2019.03.001
Database	NAL-Catalogue (AGRICOLA)

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