Development of a reverse transcription polymerase chain reaction for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals

AbstractBackgroundSevere fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble to those of SFTS patients and SFTS-contracted cats shows high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals.Methodology/Principle findingsFour primer sets were newly designed from consensus sequences constructed by 108 strains of SFTSV. A reverse transcription polymerase chain reaction (RT-PCR) with these four primer sets were successfully and specifically detected several clades of SFTSV. Their limits of detection are 1-10 copies/reaction. By this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients.Conclusion/SignificanceThis newly developed RT-PCR could detect SFTSV RNA of several clades from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.Author summaryThis study developed RT-PCR to detect SFTS animal cases. This assay could detect SFTSV RNA belonging to different clades. Cats diagnosed as SFTS had IgM or IgG, but not neutralizing antibodies. SFTS cat cases were distributed in the area where SFTS patients have been reported highly, indicating the establishment of the circulation of SFTSV in the environment. These diagnostic assays could be helpful tools to detect and not to miss SFTS animal cases.

Download Full-text

Clinical Usefulness of Nested Reverse-Transcription Polymerase Chain Reaction for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.21-0183 ◽

2021 ◽

Author(s):

Choon Mee Kim ◽

Dong-Min Kim ◽

Na-Ra Yun

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Immunofluorescence Assay ◽

Clinical Usefulness ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Pcr Targeting ◽

Severe Fever

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) is an emerging tick-borne infectious disease. Few studies have assessed the clinical usefulness of nested reverse-transcription polymerase chain reaction (RT-PCR) for diagnosing SFTS. We performed conventional RT-PCR targeting the M segment, nested RT-PCR targeting M and S segments, and real-time RT-PCR targeting the S segment of SFTSV for four patients with suspected SFTS. Although conventional RT-PCR results for the first two patients were negative at admission, nested RT-PCR using the S or M targets was positive for the same samples. Likewise, in the other two patients, initial samples were confirmed positive in all three tests, but follow-up testing demonstrated negative conventional RT-PCR and positive nested RT-PCR results. Thus, delayed testing using conventional RT-PCR or real-time RT-PCR in symptomatic patients with SFTS may result in missed diagnoses, and compared with these methods, nested RT-PCR may increase the window for obtaining positive SFTSV PCR results. Meanwhile, the indirect immunofluorescence assay showed seroconversion to SFTSV antibodies in all four patients. Nested RT-PCR for SFTSV M and S segments could help diagnose SFTS in patients testing negative by conventional RT-PCR.

Download Full-text

Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction

Phytopathology ◽

10.1094/phyto-96-0320 ◽

2006 ◽

Vol 96 (3) ◽

pp. 320-325 ◽

Cited By ~ 27

Author(s):

Nieves Capote ◽

M. Teresa Gorris ◽

M. Carmen Martínez ◽

Margarita Asensio ◽

Antonio Olmos ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Monoclonal Antibodies ◽

Real Time ◽

Reverse Transcription ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

Chain Reaction ◽

Japanese Plum ◽

Polymerase Chain

The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3′ minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected.

Download Full-text

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals

PLoS ONE ◽

10.1371/journal.pone.0238671 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0238671

Author(s):

Eun-sil Park ◽

Osamu Fujita ◽

Masanobu Kimura ◽

Akitoyo Hotta ◽

Koichi Imaoka ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Clinical Symptoms ◽

Diagnostic System ◽

Hemorrhagic Fever ◽

Laboratory Diagnosis ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Neutralization Titer ◽

Primer Sets ◽

Severe Fever

Background Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions. Methodology/principle findings Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1–10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients. Conclusion/significance This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.

Download Full-text

A Comparison Between Serological and Biological Assays in Detecting Grapevine Leafroll Associated Viruses

Plant Disease ◽

10.1094/pdis.1997.81.7.799 ◽

1997 ◽

Vol 81 (7) ◽

pp. 799-801 ◽

Cited By ~ 28

Author(s):

Adib Rowhani ◽

Jerry K. Uyemoto ◽

Deborah A. Golino

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Multiple Infections ◽

Enzyme Linked Immunosorbent Assay ◽

Vitis Vinifera L ◽

Rt Pcr ◽

Chain Reaction ◽

Biological Assays ◽

Cabernet Franc ◽

Polymerase Chain

The efficacy of the serological procedure enzyme-linked immunosorbent assay (ELISA) for detecting grapevine leafroll associated viruses (GLRaV types -1, -2, -3, and -4) was compared with indexing on Vitis vinifera L. cv. Cabernet Franc. Results of the biological assays confirmed the infectious nature of all grapevine sources testing positive by ELISA for GLRaV-1 (9 sources), GLRaV-2 (14 sources), and GLRaV-4 (14 sources), and the noninfectious nature of ELISA-negative grapevines (75 sources). However, among 57 sources testing positive by ELISA for GLRaV-3, or 24 sources with multiple infections, 8 and 1 sources, respectively, were negative by Cabernet Franc assays. Serological assays were repeated on all graft-inoculated indicators and only symptomatic ones reacted positively. Also, the 8 original GLRaV-3 sources that had tested positive by ELISA and negative by bioassay were found positive using immuno-capture/reverse transcription-polymerase chain reaction (IC/RT-PCR). The single multiple-infected source was not available for retesting. The distribution of GLRaV in infected grapevines was tested by assaying 20 to 40 samples per source of 36 plants infected with GLRaV-1, -2, -3, or -4. The incidence of GLRaV-positive canes as determined by ELISA ranged from 0 to 100%, suggesting that GLRaV can be unevenly distributed in chronically infected grapevines.

Download Full-text

Sensitive Method for Testing Peanut Seed Lots for Peanut stripe and Peanut mottle viruses by Immunocapture-Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽

10.1094/pdis.2000.84.5.559 ◽

2000 ◽

Vol 84 (5) ◽

pp. 559-561 ◽

Cited By ~ 15

Author(s):

A. G. Gillaspie ◽

R. N. Pittman ◽

D. L. Pinnow ◽

B. G. Cassidy

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Capsid Region ◽

Large Numbers ◽

Peanut Stripe Virus ◽

Pcr Method ◽

Polymerase Chain

An immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) method was developed for testing peanut (Arachis hypogaea) seed lots for infection by Peanut stripe virus (PStV) and Peanut mottle virus (PeMV). A small slice was removed from each seed distal to the radicle of a random 100-seed sample, the slices were extracted in buffer and centrifuged, and a portion of the supernatant was incubated in a tube that had been coated with antiserum to either PStV or PeMV. Following immunocapture of the virus, the tube was washed, the RT-PCR mix (with primers designed from conserved sequences within the capsid region of each virus) was placed in the same tubes, and the test completed. Results obtained on 15 previously untested seed lots from the collection indicated good correlation between virus detected by the IC-RT-PCR method and virus detected from the same seed lots by enzyme-linked immunosorbent assay (ELISA). The IC-RT-PCR method detected three lots infected with PeMV and none with PStV from 106 seed lots grown in Ecuador (results confirmed by ELISA). The IC-RT-PCR method is more sensitive than ELISA (currently used on samples consisting of five seeds), is useful for testing large numbers of seed lots of peanut germ plasm, and could be adapted to test other plants and detect other viruses.

Download Full-text

Detection and Isolate Differentiation of Citrus tristeza virus in Infected Field Trees Based on Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽

10.1094/pdis.2004.88.6.625 ◽

2004 ◽

Vol 88 (6) ◽

pp. 625-629 ◽

Cited By ~ 33

Author(s):

Zhipeng Huang ◽

Phyllis A. Rundell ◽

Xiong Guan ◽

Charles A. Powell

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Citrus Tristeza Virus ◽

Sweet Orange ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Tristeza Virus ◽

Citrus Tristeza

Reverse transcription-polymerase chain reaction (RT-PCR) was compared with enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) for detection of non-decline-inducing and decline-inducing isolates of Citrus tristeza virus (CTV) in 21 field sweet orange and grapefruit plants on sour orange rootstock in Fort Pierce, FL. Among these samples, seven, six, and eight were infected with decline-inducing, non-decline-inducing, and both decline-inducing and non-decline-inducing isolates of CTV, respectively. However, there was not a good correlation between field symptoms and detection of the decline-inducing isolate. The results confirmed that RT-PCR is not only able to detect and differentiate decline-inducing and non-decline-inducing isolates of CTV in Florida, but also can detect both isolate types in a single field sweet orange or grapefruit tree. For most samples, results from RT-PCR, ELISA, and DTBIA were the same. However, the 320-bp fragments produced only from decline-inducing isolates were amplified from two sweet orange and two grapefruit samples that did not react with decline-inducing CTV-specific monoclonal antibody MCA13 in ELISA or DTBIA, indicating that RT-PCR has a higher sensitivity than these immunological tests for field sweet orange or grapefruit samples. Thus, RT-PCR is a simple, rapid, and specific procedure for CTV identification applicable to both research and diagnostic needs.

Download Full-text

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The Journal of Urology ◽

10.1016/s0022-5347(18)33708-x ◽

2006 ◽

Vol 175 (4S) ◽

pp. 485-486

Author(s):

Sabarinath B. Nair ◽

Christodoulos Pipinikas ◽

Roger Kirby ◽

Nick Carter ◽

Christiane Fenske

Keyword(s):

Prostate Cancer ◽

Polymerase Chain Reaction ◽

Real Time ◽

Molecular Diagnosis ◽

Reverse Transcription ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain

Download Full-text

Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽

10.1159/000517003 ◽

2021 ◽

pp. 1-6

Author(s):

Salman Khan ◽

Syed Asad Ali Shah ◽

Syed Muhammad Jamal

Keyword(s):

Polymerase Chain Reaction ◽

Viral Genome ◽

Foot And Mouth Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Kappa Value ◽

Polymerase Chain ◽

Pcr Assays ◽

Level Of Agreement

Background: Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. Methods: A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. Results: S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (p = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. Conclusions: The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

Download Full-text

Expression of hyaluronan synthase-3 in porcine oviducal epithelium during oestrus

Reproduction Fertility and Development ◽

10.1071/rd02100 ◽

2003 ◽

Vol 15 (2) ◽

pp. 99 ◽

Cited By ~ 22

Author(s):

Paisan Tienthai ◽

Naoko Kimura ◽

Paraskevi Heldin ◽

Eimei Sato ◽

Heriberto Rodriguez-Martinez

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Hyaluronan Synthase ◽

Critical Periods ◽

Rt Pcr ◽

Chain Reaction ◽

Mean Values ◽

Polymerase Chain ◽

Sperm Reservoir ◽

Oviduct Fluid

Hyaluronan (HA) has been related to fertilization and embryo development in the pig. Furthermore, HA is present in pig oviduct fluid and the lining epithelium, particularly of the pre-ovulatory sperm reservoir. Because the mechanisms that regulate HA synthesis have not yet been clarified, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to assess the expression of mRNAs of two HA-synthesizing enzymes (has2 and has3) in the oviduct epithelium (uterotubal junction, isthmus, ampullary–isthmic junction and ampulla segments) of non-inseminated (control) and inseminated (treatment) sows at pre-, peri- and post-ovulatory oestrus. Only has3 mRNA was detected; it was present in all tubal segments of both control and treatment samples. The level of has3 expression did not vary significantly between non-inseminated and inseminated specimens, but there was a tendency (NS) for increased mean values during the peri- and post-ovulatory stages compared with pre-ovulation. It is concluded that has3 is expressed by the porcine endosalpinx epithelium and the levels of expression do not vary during the critical periods of sperm transport and fertilization, despite fluctuating levels of HA in the tubal fluid at corresponding periods.

Download Full-text