scholarly journals Development of an Indirect ELISA and Dot-Blot Assay for Serological Detection of Rice Tungro Disease

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Magdline Sia Henry Sum ◽  
Siew Fung Yee ◽  
Lily Eng ◽  
Evenni Poili ◽  
Julia Lamdin
Keyword(s):  
Sensitivity And Specificity ◽  
Rural Areas ◽  
Indirect Elisa ◽  
High Sensitivity ◽  
Visual Observation ◽  
Alternative Methods ◽  
Dot Blot ◽  
Specificity And Sensitivity ◽  
Rice Tungro Disease ◽  
Dot Blot Assay

Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.

scholarly journals Generation and diagnostic application of monoclonal antibodies against Seneca Valley virus

2011 ◽  
Vol 24 (1) ◽  
pp. 42-50 ◽  
Author(s):  
Ming Yang ◽  
Rebekah van Bruggen ◽  
Wanhong Xu
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Elisa ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Specific Antibody Response ◽  
Dot Blot Assay ◽  
Infected Cells ◽  
Seneca Valley Virus ◽  
Vesicular Disease

Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

scholarly journals INDIRECT ELISA FOR DETECTION OF ANTIBODIES TO FMDV NON-STRUCTURAL PROTEINS IN PORCINE SERA

2018 ◽  
pp. 13-20
Author(s):  
A. S. Yakovleva ◽  
A. V. Kanshina ◽  
A. V. Scherbakov
Keyword(s):  
Indirect Elisa ◽  
High Sensitivity ◽  
High Specificity ◽  
Structural Proteins ◽  
Post Inoculation ◽  
Nonstructural Proteins ◽  
Diagnostic Specificity ◽  
Fmd Virus ◽  
Specificity And Sensitivity ◽  
Sensitivity Specificity

An indirect variant of ELISA used for detection of antibodies to nonstructural proteins of the FMD virus in porcine blood sera was developed. The results of the validation showed that the developed method is characterized by high sensitivity, specificity and reproducibility. When testing the blood serum panel obtained from experimentally infected animals, the method allowed to detect antibodies to FMD virus in 7 of 18 sera collected on day 6 post inoculation, in 13 of 19 sera – on day 7 post inoculation, in 16 of 19 sera – on day 8 post inoculation and in all 76 sera obtained on days 9–12 post inoculation. The diagnostic specificity of 3AB-ELISA was 100% when testing 100 knowingly negative blood sera from pigs imported to Russia from Norway. High specificity and sensitivity of the method, established during the development of the method, are confirmed in the course of routine diagnostic tests.

scholarly journals Experimental use of a Dot-Blot Assay to Measure Serologic Responses of Cattle Vaccinated with Brucella Abortus Strain RB51

1997 ◽  
Vol 9 (4) ◽  
pp. 363-367 ◽  
Author(s):  
S. C. Olsen ◽  
M. G. Stevens ◽  
N. F. Cheville ◽  
G. Schurig
Keyword(s):  
Brucella Abortus ◽  
The United States ◽  
United States Department ◽  
Dot Blot ◽  
Department Of Agriculture ◽  
Specificity And Sensitivity ◽  
Colony Forming Units ◽  
Dot Blot Assay ◽  
Inspection Service ◽  
Experimental Use

Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture. Currently available serologic surveillance tests for B. abortus do not detect seroconversion following SRB51 vaccination. The purpose of this study was to evaluate a dot-blot assay using γ-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51. Dot-blot titers of sera at a recommended dosage (1010 colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B. abortus strain 19 and greater ( P < 0.05) than titers of nonvaccinated cattle. In the first 12 weeks after vaccination with 1010 colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater. Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 1010 colony-forming units of strain RB51. Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (109 colony-forming units) did not differ ( P > 0.05) from titers of sera from nonvaccinated cattle. Following intraconjunctival challenge with B. abortus strain 2308, titers on the RB51 dot-blot assay did not differ ( P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.

scholarly journals Field Validation of the Use of RB51 as Antigen in a Complement Fixation Test To Identify Calves Vaccinated withBrucella abortus RB51

2001 ◽  
Vol 8 (2) ◽  
pp. 385-387 ◽  
Author(s):  
Rosanna Adone ◽  
Franco Ciuchini ◽  
Steven Olsen
Keyword(s):  
Complement Fixation ◽  
High Specificity ◽  
Antibody Responses ◽  
Test Results ◽  
Dot Blot ◽  
Serum Samples ◽  
Specificity And Sensitivity ◽  
Routine Surveillance ◽  
Dot Blot Assay ◽  
Smooth Strain

ABSTRACT In order to confirm the efficiency of an experimental RB51-based complement fixation (CF) test in identifying cattle vaccinated withBrucella abortus strain RB51, 831 sera from 110 vaccinated and 48 unvaccinated Hereford heifers of Iowa, collected for studies conducted in different years, were sent to Italy without coding to be tested in a CF test using RB51 as antigen. Most of the calves, aged from 3 to 10 months, were vaccinated subcutaneously with the recommended dosage of 1010 CFU of RB51 commercial vaccine, while only six calves received 109 CFU of the same vaccine. Serum samples for serologic testing, collected until 16 postinoculation weeks (PIW), were also tested by routine surveillance tests for brucellosis such as rose bengal plate and CF tests performed withB. abortus smooth strain 99 as control antigen. RB51 CF test results obtained by testing sera from cattle vaccinated in 1999 indicate that the sensitivity of the reaction is 97% at 2 to 3 PIW and 90% until 8 PIW and decreases to 65% at 12 PIW, the specificity remaining at 100%. Collectively, the results of this study confirm that serologic standard tests fail to detect antibodies to RB51 while the RB51-based CF test is able to monitor antibody responses to RB51 until 15 to 16 PIW with a specificity of 100%. In addition, unlike the RB51-based dot blot assay, which is the only test currently used to monitor antibody responses to RB51, the CF test also detected specific responses following vaccination with 109 CFU of RB51, although seroconversion was only 50% at 8 PIW. In conclusion, because of high specificity and sensitivity, the CF test described here can be used to efficaciously monitor serologic responses following RB51 vaccination in cattle and could also be employed to detect RB51 infection in humans exposed to this strain.

scholarly journals Comparison between molecular methods (PCR vs LAMP) to detect Candida albicans in bronchoalveolar lavage samples of suspected tuberculosis patients

2018 ◽  
Vol 8 (2) ◽  
Author(s):  
Meysam Goodarzi ◽  
Mohammad Hassan Shahhosseiny ◽  
Mansour Bayat ◽  
Seyed Jamal Hashemi ◽  
Mohammad Ghahri
Keyword(s):  
Bronchoalveolar Lavage ◽  
Sensitivity And Specificity ◽  
Isothermal Condition ◽  
High Sensitivity ◽  
High Specificity ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Specificity And Sensitivity ◽  
Suspected Tuberculosis ◽  
Pcr Test

With the increase of patients suffering from immune deficiency infections also increased pulmonary fungi even in people with defective immune system can cause fatal and lethal candidiasis. The timely diagnosis of pulmonary candidiasis is one of the problems that has been detected. Polymerase chain reaction (PCR) test and Loop mediated isothermal amplification (LAMP) method optimized on the basis of α INT1 gene and then sensitivity and specificity were evaluated. LAMP is a novel nucleic acid amplification technique with high specificity and sensitivity which has been done under isothermal condition. Samples were the bronchoalveolar lavage suspected of tuberculosis (TB) reviews for TB disease negative have been reported. DNA extraction carried out by standard phenol/chloroform method on samples and PCR test and LAMP was done. PCR and LAMP testing was performed on samples and products of 441 bp were amplified and observed with agarose gel electrophoresis. At the end of the LAMP reaction, SYBR Green was used for identifying negative and positive results. Among the 60 quantities sera, only 7 cases were PCR positive but 8 cases were LAMP positive. In comparison, between LAMP and PCR, the LAMP technique in spite of its simplicity, high sensitivity and specificity, could be an appropriate replacement for PCR.

scholarly journals Transmembrane and Tetratricopeptide Repeat Containing 4 Is a Novel Diagnostic Marker for Prostate Cancer with High Specificity and Sensitivity

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1029
Author(s):  
Rania Makboul ◽  
Islam F. Abdelkawi ◽  
Dalia M. Badary ◽  
Mahmoud R. A. Hussein ◽  
Johng S. Rhim ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
High Specificity ◽  
Normal Prostate ◽  
Protein Levels ◽  
Prostate Epithelial Cells ◽  
Specificity And Sensitivity ◽  
Histopathologic Diagnosis ◽  
Tissue Specimens

The histopathologic diagnosis of prostate cancer (PCa) from biopsies is a current challenge if double or triple staining is needed. Therefore, there is an urgent need for development of a new reliable biomarker to diagnose PCa patients. We aimed to explore and compare the expression of TMTC4 in PCa cells and tissue specimens and evaluate its sensitivity and specificity. The expression of TMTC4 in PCa and normal prostate epithelial cells was determined by real-time PCR and Western blot analyses. Immunohistochemical (IHC) staining of TMTC4 was performed on tissues collected from PCa and benign prostatic hyperplasia (BPH). Our results show a high expression of TMTC4 on mRNA and protein levels in PCa versus BPH1 and normal cells (p < 0.05). IHC results show strong cytoplasmic expressions in PCa cases (p < 0.001) as compared to BPH cases. The overall accuracy as measured by the AUC was 1.0 (p < 0.001). The sensitivity and specificity of the protein were 100% and 96.6%, respectively. Taken together, we report a high TMTC4 expression in PCa cells and tissues and its ability to differentiate between PCa and BPH with high sensitivity and specificity. This finding can be carried over to clinical practice after its confirmation by further studies.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

2013 ◽  
Vol 20 (8) ◽  
pp. 1143-1149 ◽  
Author(s):  
Weiwei Yan ◽  
Muhammad Hassan Saleem ◽  
Patrick McDonough ◽  
Sean P. McDonough ◽  
Thomas J. Divers ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Serum Dilution ◽  
Content Type ◽  
Diagnostic Antigen ◽  
Repeat Domain ◽  
Associated Proteins ◽  
Dot Blot Assay

ABSTRACTLeptospiraimmunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5–5.5), 5.5th to 6.5th (LigACon5.5–6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that LigACon4-7.5 is the best candidate diagnostic antigen in a slot-shaped dot blot assay. LigACon4-7.5 was further evaluated as an indirect enzyme-linked immunosorbent assay (ELISA) antigen for the detection ofLeptospiraantibodies in equine sera. This assay was evaluated with equine sera (n= 60) that were microscopic agglutination test (MAT) negative and sera (n= 220) that were MAT positive to the 5 serovars that most commonly cause equine leptospirosis. The indirect ELISA results showed that at a single serum dilution of 1:250, the sensitivity and specificity of ELISA were 80.0% and 87.2%, respectively, compared to those of MAT. In conclusion, an indirect ELISA was developed utilizing a recombinant LigA fragment comprising the 4th to 7.5th repeat domain (LigACon4-7.5) as a diagnostic antigen for equine leptospirosis. This ELISA was found to be sensitive and specific, and it yielded results that concurred with those of the standard MAT.

scholarly journals Evaluation of Xpert GBS assay and Xpert GBS LB assay for detection of Streptococcus agalactiae

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Meng-Yi Han ◽  
Chen Xie ◽  
Qing-Qing Huang ◽  
Qiao-Hua Wu ◽  
Qing-Yun Deng ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Publication Bias ◽  
Sensitivity And Specificity ◽  
Area Under The Curve ◽  
Neonatal Morbidity ◽  
High Sensitivity ◽  
Alternative Methods ◽  
Cochrane Library ◽  
Diagnostic Standards ◽  
Group B

Abstract Background Group B Streptococcal (GBS) infection is the primary agent of neonatal morbidity and mortality. Rapid and simple methods to detect GBS are Xpert GBS and GBS LB assays based on real-time polymerase chain reaction (PCR). However, since the diagnostic accuracy of the two techniques in diagnosing GBS remains unclear, we designed this study to appraise the diagnostic accuracy of the aforementioned. Methods A systematic search of all literature published before July 16, 2020 was conducted using Embase, PubMed, Web of Science, and Cochrane Library. The study quality was evaluated through Review Manager 5.3. Accordingly, data extracted in the included studies were analyzed using Meta-DiSc 1.4 and Stata 12.0 software. The diagnosis odds ratio (DOR) and bivariate boxplot were utilized to evaluate the heterogeneity. Publication bias was appraised by using Deeks’ funnel plot. Results A total of 13 studies were adopted and only 19 sets of data met the criteria. The sensitivity and specificity of Xpert GBS were 0.91 (95% CI 0.89–0.92) and 0.93 (95% CI 0.92–0.94). The area under the curve (AUC) was 0.9806. The sensitivity and specificity results of Xpert GBS LB were 0.96 (95% CI 0.95–0.98) and 0.94 (95% CI 0.92–0.95), respectively. The AUC was 0.9950. No publication bias was found. Conclusions The Xpert GBS and GBS LB assays are valuable alternative methods with high sensitivity and specificity. However, determining whether they can be used as clinical diagnostic standards for GBS is essential for the future.

scholarly journals RAPD and ERIC-PCR coupled with HRM for species identification of non-dysenteriae Shigella species; as a potential alternative method

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Babak Pakbin ◽  
Afshin Akhondzadeh Basti ◽  
Ali Khanjari ◽  
Leila Azimi ◽  
Wolfram Manuel Brück ◽  
...  
Keyword(s):  
Species Identification ◽  
Sensitivity And Specificity ◽  
Principal Component ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Alternative Methods ◽  
Clinical Specimens ◽  
Alternative Method ◽  
Rapd Pcr ◽  
Potential Alternative

Abstract Objective Species identification of Shigella isolates are so prominent for epidemiological studies and infection prevention strategies. We developed and evaluated RAPD and ERIC-PCR coupled with HRM for differentiation of non-dysenteriae Shigella species as potential alternative methods. After isolation of eighteen Shigella strains from faecal specimens collected from children under 2 years of age with diarrhea (n = 143), the species of the isolates were identified by slide agglutination assay. Also, species were identified using developed RAPD-PCR-HRM and ERIC-PCR-HRM techniques. Differentiation of the data sets was measured by principal component analysis as a dimension reduction method. Then, sensitivity and specificity of the methods were evaluated. Results We found RAPD-PCR-HRM method with high sensitivity and specificity (100 and 85% respectively) to identify non-dysenteriae Shigella species in clinical specimens. However, sensitivity and specificity of ERIC-PCR-HRM were evaluated 33 and 46% respectively and significantly lower than that of RAPD-PCR-HRM assay. Regardless of inherent poor reproducibility of DNA fingerprinting-based methods, RAPD-PCR-HRM assay can be considered as a potential alternative method to identify non-dysenteriae species of Shigella in clinical specimens. As we observed in the current study, HRM technique is more rapid, inexpensive, and sensitive than gel electrophoresis method to characterize PCR amplicons.

scholarly journals Laboratory evaluation of the rapid diagnostic tests for the detection of Vibrio cholerae O1 using diarrheal samples

2021 ◽  
Vol 15 (6) ◽  
pp. e0009521
Author(s):  
Goutam Chowdhury ◽  
Tarosi Senapati ◽  
Bhabatosh Das ◽  
Asha Kamath ◽  
Debottam Pal ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Sensitivity And Specificity ◽  
Gold Standard ◽  
Diagnostic Tests ◽  
Diarrheal Disease ◽  
High Sensitivity ◽  
Rapid Diagnostic Tests ◽  
Laboratory Evaluation ◽  
Specificity And Sensitivity ◽  
Stool Samples

Background Cholera, an acute diarrheal disease is a major public health problem in many developing countries. Several rapid diagnostic tests (RDT) are available for the detection of cholera, but their efficacies are not compared in an endemic setting. In this study, we have compared the specificity and sensitivity of three RDT kits for the detection of Vibrio cholerae O1 and compared their efficiency with culture and polymerase chain reaction (PCR) methods. Methods Five hundred six diarrheal stool samples collected from patients from two different hospitals in Kolkata, India were tested using SD Bioline Cholera, SMART-II Cholera O1 and Crystal-VC RDT kits. All the stool samples were screened for the presence of V. cholerae by direct and enrichment culture methods. Stool DNA-based PCR assay was made to target the cholera toxin (ctxAB) and O1 somatic antigen (rfb) encoding genes. Statistical evaluation of the RDTs has been made using STATA software with stool culture and PCR results as the gold standards. The Bayesian latent class model (LCM) was used to evaluate the diagnostic tests in the absence of the gold standard. Results Involving culture technique as gold standard, the sensitivity and specificity of the cholera RDT kits in the direct testing of stools was highest with SAMRT-II (86.1%) and SD-Cholera (94.4%), respectively. The DNA based PCR assays gave very high sensitivity (98.4%) but the specificity was comparatively low (75.3%). After enrichment, the high sensitivity and specificity was detected with SAMRT-II (78.8%) and SD-Cholera (99.1%), respectively. Considering PCR as the gold standard, the sensitivity and specificity of the RDTs remained between 52.3–58.2% and 92.3–96.8%, respectively. In the LCM, the sensitivity of direct and enrichment testing was high in SAMRT-II (88% and 92%, respectively), but the specificity was high in SD cholera for both the methods (97% and 100%, respectively). The sensitivity/specificity of RDTs and direct culture have also been analyzed considering the age, gender and diarrheal disease severity of the patients. Conclusion Overall, the performance of the RDT kits remained almost similar in terms of specificity and sensitivity. Performance of PCR was superior to the antibody-based RDTs. The RTDs are very useful in identifying cholera cases during outbreak/epidemic situations and for making them as a point-of-care (POC) testing tool needs more improvement.

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