scholarly journals Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Changzhong Jin ◽  
Nanping Wu ◽  
Xiaorong Peng ◽  
Hangping Yao ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Influenza A ◽  
High Specificity ◽  
High Viral Load ◽  
Immunochromatographic Assay ◽  
Clinical Samples ◽  
H7n9 Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Viral Culture

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings.

scholarly journals Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study

1999 ◽  
Vol 37 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Britta Gröndahl ◽  
Wolfram Puppe ◽  
Andrea Hoppe ◽  
Inka Kühne ◽  
Josef A. I. Weigl ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Reverse Transcription ◽  
Influenza A ◽  
Respiratory Tract Infections ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Tract Infections ◽  
Type 3

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

scholarly journals Effectiveness of two rapid influenza tests in comparison to reverse transcription-PCR for influenza A diagnosis

2014 ◽  
Vol 8 (03) ◽  
pp. 331-338 ◽  
Author(s):  
Ramón Zazueta-García ◽  
Adrian Canizalez-Roman ◽  
Hector Flores-Villaseñor ◽  
Javier Martínez-Garcia ◽  
Alejandro Llausas-Vargas ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Correct Diagnosis ◽  
Immunofluorescence Assay ◽  
Immunochromatographic Assay ◽  
High Morbidity ◽  
Rt Pcr ◽  
Predictive Values ◽  
Reverse Transcription Pcr ◽  
Influenza A H1n1

Introduction: The influenza A virus is responsible for high morbidity and mortality in children and adults worldwide. Thus, a rapid, sensitive, and specific diagnosis tool is required. Methodology: An immunofluorescence assay (DFA) and a lateral-flow immunochromatographic assay were compared with RT-PCR for detection of the influenza A virus in 113 nasopharyngeal wash samples obtained from pediatric patients. Samples were collected between July and December 2009, during the pandemic outbreak of influenza A H1N1/09. Results: The sensitivity, specificity, and positive and negative predictive values obtained for the DFA were 68.97%, 76.63%, 75.47%, and 70%, respectively, while the values obtained for the immunochromatographic assay were 58.62%, 81.82%, 77.27%, and 65.22%, respectively. The frequency of the influenza A virus was 51.33%, and a total of 27 samples were positive for the pandemic influenza A H1N1/09. Conclusions: DFA and the immunochromatographic assay can be important tools for patient care during influenza season and in outbreaks as they usually provide results within 45 minutes. Furthermore, positive results in conjunction with the patient’s symptoms could provide a correct diagnosis, thus facilitating appropriate patient management. Nonetheless, the results of these assays still require confirmation by RT-PCR.

scholarly journals Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

scholarly journals Identification of Encephalomyocarditis Virus in Clinical Samples by Reverse Transcription-PCR Followed by Genetic Typing Using Sequence Analysis

1998 ◽  
Vol 36 (12) ◽  
pp. 3463-3467 ◽  
Author(s):  
H. Vanderhallen ◽  
F. Koenen
Keyword(s):  
Sequence Analysis ◽  
Reverse Transcription ◽  
Molecular Techniques ◽  
Encephalomyocarditis Virus ◽  
Clinical Samples ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Field Samples ◽  
Pcr Targeting

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3′ end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.

scholarly journals Reverse transcription-PCR using a Primer Set Targeting the 3D Region Detects Foot-and-Mouth Disease Virus with High Sensitivity

2018 ◽  
Author(s):  
Tatsuya Nishi ◽  
Toru Kanno ◽  
Nobuaki Shimada ◽  
Kazuki Morioka ◽  
Makoto Yamakawa ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Pcr Assays

AbstractBecause foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5’-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 101-to 104-fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

scholarly journals Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR

2000 ◽  
Vol 38 (3) ◽  
pp. 1191-1195 ◽  
Author(s):  
Jose C. Aguilar ◽  
María P. Pérez-Breña ◽  
María L. García ◽  
Nieves Cruz ◽  
Dean D. Erdman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Reverse Transcription ◽  
Pediatric Patients ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Parainfluenza Viruses ◽  
Cell Culture Isolation ◽  
Human Parainfluenza Viruses

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID50) for HPIV type 4B (HPIV-4B) to 32 TCID50s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.

scholarly journals Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

2001 ◽  
Vol 67 (12) ◽  
pp. 5593-5600 ◽  
Author(s):  
Julie Jean ◽  
Burton Blais ◽  
André Darveau ◽  
Ismaı̈l Fliss
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Dot Blot ◽  
Reverse Transcription Pcr ◽  
Dot Blot Assay

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

scholarly journals Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza A+B Test for Rapid Diagnosis of Influenza

2007 ◽  
Vol 45 (4) ◽  
pp. 1234-1237 ◽  
Author(s):  
M. Mehlmann ◽  
A. B. Bonner ◽  
J. V. Williams ◽  
D. M. Dankbar ◽  
C. L. Moore ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Influenza A ◽  
Rapid Diagnosis ◽  
Reverse Transcription Pcr ◽  
Viral Culture ◽  
B Test

scholarly journals The evaluation of a novel digital immunochromatographic assay with silver amplification to detect SARS-CoV-2

2021 ◽  
Author(s):  
Yoko Kurihara ◽  
Yoshihiko Kiyasu ◽  
Yusaku Akashi ◽  
Yuto Takeuchi ◽  
Kenji Narahara ◽  
...  
Keyword(s):  
Predictive Value ◽  
Limit Of Detection ◽  
The Other ◽  
Nucleic Acid Amplification ◽  
Immunochromatographic Assay ◽  
Clinical Samples ◽  
Antigen Test ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Antigen Tests

Introduction Rapid antigen tests are convenient for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they have lower sensitivities than nucleic acid amplification tests. In this study, we evaluated the diagnostic performance of Quick Chaser Auto SARS-CoV-2, a novel digital immunochromatographic assay that is expected to have higher sensitivity than conventional antigen tests. Methods A prospective observational study was conducted between February 8 and March 24, 2021. We simultaneously obtained two nasopharyngeal samples, one for evaluation with the QuickChaser Auto SARS-CoV-2 antigen test and the other for assessment with reverse transcription PCR (RT-PCR), considered the gold-standard reference test. The limit of detection (LOD) of the new antigen test was compared with those of four other commercially available rapid antigen tests. Results A total of 1401 samples were analyzed. SARS-CoV-2 was detected by reference RT-PCR in 83 (5.9%) samples, of which 36 (43.4%) were collected from symptomatic patients. The sensitivity, specificity, positive predictive value, and negative predictive value were 74.7% (95% confidence interval (CI): 64.0-83.6%), 99.8% (95% CI: 99.5-100%), 96.9% (95% CI: 89.2-99.6%), and 98.4% (95% CI: 97.6-99.0%), respectively. When limited to samples with a cycle threshold (Ct) <30 or those from symptomatic patients, the sensitivity increased to 98.3% and 88.9%, respectively. The QuickChaser Auto SARS-CoV-2 detected 34-120 copies/test, which indicated greater sensitivity than the other rapid antigen tests. Conclusions QuickChaser Auto SARS-CoV-2 showed sufficient sensitivity and specificity in clinical samples of symptomatic patients. The sensitivity was comparable to RT-PCR in samples with Ct<30.

scholarly journals Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

2016 ◽  
Vol 55 (3) ◽  
pp. 759-767 ◽  
Author(s):  
Japhette Esther Kembou Tsofack ◽  
Rachel Zamostiano ◽  
Salsabeel Watted ◽  
Asaf Berkowitz ◽  
Ezra Rosenbluth ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Samples ◽  
Fish Cell ◽  
Rt Pcr ◽  
Fish Cell Lines ◽  
Pcr Assay ◽  
Farmed Fish ◽  
Reverse Transcription Pcr ◽  
Aquaculture Industry ◽  
First Time

ABSTRACT Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

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