scholarly journals Detection of human enteric viruses in stream water with RT-PCR and cell culture

2004 ◽  
Vol 2 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Kimberly Denis-Mize ◽  
G. Shay Fout ◽  
Daniel R. Dahling ◽  
Donna S. Francy
Keyword(s):  
Cell Culture ◽  
Membrane Filtration ◽  
Stream Water ◽  
False Negative ◽  
Molecular Data ◽  
Sampling Procedure ◽  
Rt Pcr ◽  
Quality Controls ◽  
Pcr Method ◽  
Virus Recovery

A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

scholarly journals The PI3KCA and AKT Inhibitory Activities of Litsea Cubeba Lour. Fruits and Heartwoods Towards Hela Cells

2019 ◽  
Vol 7 (9) ◽  
pp. 1422-1424
Author(s):  
Aminah Dalimunthe ◽  
Poppy Anjelisa Zaitun Hasibuan ◽  
Denny Satria
Keyword(s):  
Gene Expression ◽  
Cervical Cancer ◽  
Cell Culture ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Litsea Cubeba ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Inhibitory Activities

AIM: To investigated the activities of chloroform fractions at pH 7 of Litsea cubeba Lour. Fruits and heartwoods (CF-7F and CF-7H) in decrease expression of PI3KCA, Akt-1 and Akt-2 genes towards cervical cancer cell culture (HeLa) experiments in vitro. MATERIAL AND METHODS: CF-7F and CF-7H (12.5 and 25 µg/mL) were tested for its potential inhibition on gene expression of PI3KCA, Akt-1 and Akt-2 genes by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. RESULT: CF-7F and CF-7H were showed the activity to reduce the expression of PI3KCA, Akt-1 and Akt-2 genes. CONCLUSION: Our results suggest that CF-7F and CF-7H significantly inhibit the expression of PI3KCA, Akt-1 and Akt-2 genes.

Efficiency of MS2 phage and Qβ phage removal by membrane filtration in water treatment: Applicability of real-time RT-PCR method

2009 ◽  
Vol 326 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Jérémie Langlet ◽  
Leslie Ogorzaly ◽  
Jean-Christophe Schrotter ◽  
Claire Machinal ◽  
Fabien Gaboriaud ◽  
...  
Keyword(s):  
Water Treatment ◽  
Real Time ◽  
Membrane Filtration ◽  
Rt Pcr ◽  
Pcr Method

scholarly journals Detection of Astroviruses, Enteroviruses, and Adenovirus Types 40 and 41 in Surface Waters Collected and Evaluated by the Information Collection Rule and an Integrated Cell Culture-Nested PCR Procedure

2000 ◽  
Vol 66 (6) ◽  
pp. 2520-2525 ◽  
Author(s):  
Christopher D. Chapron ◽  
Nicola A. Ballester ◽  
Justin H. Fontaine ◽  
Christine N. Frades ◽  
Aaron B. Margolin
Keyword(s):  
Cell Culture ◽  
Surface Water ◽  
Cell Line ◽  
Water Samples ◽  
Nested Pcr ◽  
Information Collection ◽  
Rt Pcr ◽  
Human Astroviruses ◽  
Pcr Method ◽  
Surface Water Samples

ABSTRACT We evaluated the use of an integrated cell culture-reverse transcription-PCR (ICC-RT-PCR) procedure coupled with nested PCR to detect human astroviruses, enteroviruses, and adenovirus types 40 and 41 in surface water samples that were collected and evaluated by using the Information Collection Rule (ICR) method. The results obtained with the ICC-RT-PCR–nested PCR method were compared to the results obtained with the total culturable virus assay–most-probable-number (TCVA-MPN) method, the method recommended by the U.S. Environmental Protection Agency for monitoring viruses in surface and finished waters. Twenty-nine ICR surface water samples were analyzed. Viruses were concentrated by using filter adsorption-beef extract elution and organic flocculation techniques, and then the preparations were evaluated for viruses by visualizing cytopathic effects in the Buffalo green monkey kidney (BGMK) cell line. In the ICC-RT-PCR–nested PCR technique we used Caco-2 cells to propagate astroviruses and enteroviruses (ICC step), and we used BGMK cells to propagate adenovirus types 40 and 41, as well as enteroviruses. Fifteen of the 29 samples (51.7%) were positive for astrovirus as determined by the ICC-RT-PCR–nested PCR method, and eight of these samples (27.5%) contained infectious astrovirus. Seventeen of the 29 samples (58.6%) were positive for enteroviruses when the BGMK cell line was used, and six (27.6%) of these samples were determined to be infectious. Fourteen of the 29 samples (48.3%) were positive for adenovirus types 40 and 41, and 11 (37.9%) of these samples were determined to be infectious. Twenty-seven of the 29 samples (93.1%) were positive for a virus, and 19 (68.9%) of the samples were positive for an infectious virus. Only 5 of the 29 samples (17.2%) were positive as determined by the TCVA-MPN method. The ICC-RT-PCR–nested PCR method provided increased sensitivity compared to the TCVA-MPN method.

scholarly journals Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N2, RdRP and human RP genes

2021 ◽  
Author(s):  
Huseyin Tombuloglu ◽  
Hussein Sabit ◽  
Ebtesam Al-Suhaimi ◽  
Hamoud Al-Khallaf ◽  
Juma Kabanja ◽  
...  
Keyword(s):  
Detection Method ◽  
Limit Of Detection ◽  
Virus Disease ◽  
False Negative ◽  
Rt Pcr ◽  
Medical Centers ◽  
Efficient Control ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Commercial Kits

Abstract Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative accuracy, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, some of these assays have the disadvantages of taking time to show the result and might produce false-negative and false-positive ones. Therefore, designing rapid and accurate PCR-based testing assay is of paramount importance for early detection of this virus and for more efficient control of the spread of this disease. We, here, describe a fast, reliable, easy-to- use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N2 and RdRP) and a human gene (RP) simultaneously. The performance and the accuracy of the assay was tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.25 copies/µL or 5 copies/reaction. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Optimisation of the PEG reconcentration procedure for virus detection by cell culture or genomic amplification

1997 ◽  
Vol 35 (11-12) ◽  
pp. 455-459 ◽  
Author(s):  
Ph. Vilaginès ◽  
A. Suarez ◽  
B. Sarrette ◽  
R. Vilaginès
Keyword(s):  
Cell Culture ◽  
Virus Detection ◽  
Second Step ◽  
Virus Concentration ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Field Samples ◽  
Group A ◽  
Virus Recovery ◽  
Higher Sensitivity

A double reconcentration procedure was developed for virus detection in tapwater concentrates obtained by conventional adsorption-elution techniques suitable for cell inoculation as well as for genomic amplification. Using 7.5% PEG 6000 and 2.5% NaCl, a 15min contact time under agitation at room temperature followed by centrifugation (first step: 3,500xg, 90min, 4°C; second step 10,000xg, 20min, 4°C) were the conditions to obtain overall average virus recovery efficiencies of 71% for poliovirus from 900ml eluates and 88, 83 and 75% for poliovirus, coxsackie B2 and rotavirus respectively (400ml eluates). Direct extraction of viral RNA from the first PEG pellet with TrizolTM was efficient for RT-PCR assays without any further treatment. Primer pairs were selected to amplify rotavirus group A and poliovirus in seeded tapwater concentrated by adsorption elution through glass wool. A positive signal was obtained for theoretic virus concentration of 1 PFU. Analysis of field samples (1001) by cell culture and genomic amplification resulted in a higher sensitivity with the latter.

scholarly journals Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens

1998 ◽  
Vol 36 (6) ◽  
pp. 1741-1745 ◽  
Author(s):  
Francisco Pozo ◽  
Inmaculada Casas ◽  
Antonio Tenorio ◽  
Gloria Trallero ◽  
Jose M. Echevarria
Keyword(s):  
Cell Culture ◽  
Cerebrospinal Fluid ◽  
Aseptic Meningitis ◽  
Reverse Transcription ◽  
Diagnostic Systems ◽  
Rt Pcr ◽  
Enteroviral Infection ◽  
Reverse Transcription Pcr ◽  
Sporadic Cases ◽  
Pcr Method

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.

scholarly journals RT-PCR based detection and adaptation of foot and mouth disease virus serotype “A” in BHK-21 cell line

2016 ◽  
Vol 27 (1) ◽  
pp. 64-69
Author(s):  
MR Quddus ◽  
ML Hossen ◽  
MMR Chowdhury ◽  
T Chakrobarty ◽  
S Mahmud ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Foot And Mouth Disease ◽  
Culture Fluid ◽  
Mouth Disease ◽  
Rt Pcr ◽  
Serotype A ◽  
Foot And Mouth ◽  
Cell Culture Fluid ◽  
Pcr Method

The economic consequences of an outbreak of Foot and Mouth Disease (FMD) in Bangladesh is devastating. A sensitive, reliable and rapid diagnosis is crucial for the effective control of FMD. The present research was conducted for quick molecular detection and adaptation of FMD virus (FMDV) in BHK-21 (Baby Hamster Kidney) cell line. A two-step RT-PCR method was applied for the detection of the FMDV. Without prior adaption into BHK-21 cell culture, it is difficult to detect FMDV directly from the field samples by RT-PCR method. Samples were collected from the tongue epithelium (N=19) and inter digital tissues (N=4) of the suspected animals in Sujanagar, Bera, Santhia of Pabna district and Kotowali, Trishal of Mymensingh districts of Bangladesh during the year 2014. BHK-21 cell subculture was done from a previously cultured bottle containing BHK-21 cells. Prepared inocula were inoculated into BHK-21 cell culture and incubated at 37ºC for 24 h. After 36 h, cytopathic effects (CPE) were observed in BHK-21 cell line characterized by rounding and flattening of the cells, multinucleated giant cells formation, breaking down of the intracellular bridges and finally cell death indicated the presence of FMDV. Clear infectious BHK-21 cell culture fluid was collected and preserved at -20ºC temperature for virus detection by RT-PCR with serotype specific primers. Viral RNA was extracted from the clear infectious cell culture fluid for cDNA synthesis and used for PCR. Out of 23 samples tested, 3(13.04%) were positive for FMDV serotype A. The findings of this study can be helpful for the selection of vaccine having specific FMDV type, and it may help in controlling FMD in Bangladesh.Progressive Agriculture 27 (1): 64-69, 2016

A combined cell-culture and RT-PCR method for rapid detection of piscine nodaviruses

2001 ◽  
Vol 24 (4) ◽  
pp. 231-236 ◽  
Author(s):  
T Iwamoto ◽  
K Mori ◽  
M Arimoto ◽  
T Nakai
Keyword(s):  
Cell Culture ◽  
Rapid Detection ◽  
Rt Pcr ◽  
Pcr Method

scholarly journals Possibility for use of RT-PCR technique in establishing presence of bovine viral diarrhea virus in sperm of breeding bulls

2005 ◽  
Vol 59 (3-4) ◽  
pp. 371-381
Author(s):  
Tamas Petrovic ◽  
Sava Lazic ◽  
Milovan Jovicin ◽  
Bosiljka Djuricic
Keyword(s):  
Cell Culture ◽  
Microtiter Plate ◽  
Bovine Viral Diarrhea ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Culture Cells ◽  
Three Samples ◽  
Pcr Method ◽  
Set Up

The bovine viral diarrhea (BVD) virus is a significant health-economic pathogen in cattle which can be excreted and spread also through sperm of persistently or acutely infected bulls. Native sperm of 6 bulls, found to be negative to the BVD virus by isolating the virus and using the RT-PCR method, was experimentally infected with a tenfold dilution of the non-cytopathogen 22146 strain of the BVD virus with a titer of 105,5. This way, dilutions of the BVD virus from 10-1 to 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 in 0.1 ml native sperm were obtained. From sperm infected in this way, the virus was reisolated on FTB cell culture in a microtiter plate with 96 pools in which each sample of the infected sperm was set up in three samples, and each of them was titrated to a dilution of 1:2 to 1:256. The presence of the BVD virus was proven using the technique of fluorescent antibodies in a second blind passage on FTB culture cells. For cell culture an extremely toxic effect of native sperm to a dilution of 1:64 was established. The BVD virus was reisolated from sperm in all three sperm samples with 5 x 104, 5 x 103 i 5 x 102 TCID/50, and it was not reisolated from sperm with 50, with 5, and with 0.5 TCID/50 BVD virus in 0.1 ml native sperm. At the same time, the presence of the BVD viral genome was proved using the RT-PCR method in the same samples of artificially infected native sperm of bulls. A positive re suit was established in native sperm with 5 x 104, 5 x 103, 5 x 102 and 50 TCID/50 BVD virus n 0.1 ml native sperm. The experiment proved that the RT-PCR method has advantages over the isolation of the BVD virus from samples of native sperm of bulls. These are: shortterm investigations (1 to 2 days) and greater sensitivity (10 times bigger than the isolation of the virus). The isolation of the virus takes at least 10 days, and its greater sensitivity is primarily a result of the cyrotoxic effect of native sperm of bulls on cell culture.

Development of an astrovirus RT–PCR detection assay for use with conventional, real-time, and integrated cell culture/RT–PCR

2004 ◽  
Vol 50 (4) ◽  
pp. 269-278 ◽  
Author(s):  
Ann C Grimm ◽  
Jennifer L Cashdollar ◽  
Frederick P Williams ◽  
G Shay Fout
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Positive Control ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Detection Assay ◽  
Stool Samples ◽  
Pcr Method

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription – polymerase chain reaction (RT–PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT–PCR (ICC/RT–PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT–PCR assay was compared with RT–PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.Key words: astrovirus, RT–PCR, real-time PCR, ICC/RT–PCR, environmental water.

Sign in / Sign up

Export Citation Format

Share Document