Extraction of norovirus based on nanomagnetic beads and establishment of a fluorescence quantitative detection method

2020 ◽  
Vol 10 (9) ◽  
pp. 1463-1469
Author(s):  
Hui Chen ◽  
Kai Liu ◽  
Ziqi Xiao ◽  
Shaoyong Chen ◽  
Hang Liu ◽  
...  
Keyword(s):  
Acute Gastroenteritis ◽  
Detection Method ◽  
Melting Curve ◽  
Reaction System ◽  
Threshold Value ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Effective Prevention ◽  
Positive Stool ◽  
Pcr Method

Norovirus infection is the main cause of epidemic acute gastroenteritis outbreaks. As a result, norovirus merits careful attention. Acute gastroenteritis caused by norovirus is very harmful, necessitating effective prevention and monitoring. This study extracted norovirus using the Fe3O4 nanomagnetic bead method while establishing a rapid and sensitive quantitative norovirus detection method for emergency use. Norovirus RNA was extracted from norovirus-positive stool samples, norovirus-specific primers were designed, and SYBR Green I fluorescent dye was used to construct specific fluorescence quantitative reverse-transcription polymerase chain reaction (RT-PCR) method. Norovirus in the sample was successfully detected with the quantitative RT-PCR method established in this study, with a measured cycle threshold value of 28.73; the melting temperature value of the PCR product was approximately 83.5 °C. Additionally, by analyzing melting curve with quantitative PCR and evaluating the repeatability of the detection method, the performance of the reaction system in terms of specificity and repeatability was determined to be good. The fluorescence quantitative detection method established here can rapidly and effectively detect norovirus, and the method has good specificity and high repeatability. Currently, there is a trend of norovirus infection becoming increasingly severe. Thus, this rapid and sensitive fluorescence quantitative detection method will play a significant role in the monitoring and detection of norovirus and is thus worthy of promotion and application.

scholarly journals Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water

2003 ◽  
Vol 69 (10) ◽  
pp. 6311-6315 ◽  
Author(s):  
Miguel Angel Jiménez-Clavero ◽  
Carlos Fernández ◽  
José Antonio Ortiz ◽  
Javier Pro ◽  
Gregoria Carbonell ◽  
...  
Keyword(s):  
Water Contamination ◽  
Animal Species ◽  
Detection Method ◽  
Quantitative Detection ◽  
Pig Slurry ◽  
Rt Pcr ◽  
Rna Detection ◽  
Detection Assay ◽  
Pcr Method ◽  
Porcine Origin

ABSTRACT Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (n = 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination.

scholarly journals The Multiple Infections of Porcine Diarrhea Viruses in Local Area Based on A Luminex xTAG Multiplex Detection Method

2020 ◽  
Author(s):  
Huili liu ◽  
Ying Shi ◽  
Benqiang Li ◽  
Jie Tao ◽  
Jinghua Cheng
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Local Area ◽  
Clinical Samples ◽  
Multiple Infections ◽  
Quantitative Detection ◽  
Multiplex Detection ◽  
Viral Diarrhea ◽  
Infection Pattern ◽  
Pcr Method

Abstract Background Porcine viral diarrhea can cause great damage to the pig industry. However, multiple infections have contributed to the poor control of diarrhea, which has also resulted in great difficulties in determining the main pathogenic factors. Methods A Luminex xTAG multiplex detection method was developed for the detection of 11 viral diarrhea pathogens, which allows for the simultaneous qualitative and quantitative detection of viral diarrhea pathogens in clinical samples. A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017, and the pathogen spectrums and co-infections were analyzed. Results The minimum detection rate of the Luminex xTAG multiplex detection method was at least 10 times higher than the traditional PCR method. Of the 518 diarrhea samples, PEDV was found in 17.57% (91/518), PKoV in 40.35% (209/518), PAstV in 26.64% (138/518), PSV in 15.06% (78/518), PoSaV in 13.13% (68/518), PTV in 5.21% (27/518), PDCoV in 4.83% (25/518), PoRV in 3.28% (17/518), TGEV in 3.09% (16/518), PToV in 1.93% (10/518), and BVDV in 1.74% (9/518), respectively. Furthermore, multiple infections were commonly seen, with positive rate of 35.14%. Infection pattern of the viral diarrheal pathogens in a specific farm was changing, and different farms had the various diarrhea infection patterns. A longitudinal investigation showed that PEDV was still the key pathogen which was closely related to the death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease. Conclusions Here we provided a Luminex xTAG multiple detection method for viral diarrhea pathogen infection in clinical, which was more sensitive and specific than general multiplex PCR method. Furthermore, the surveillance confirmed high infection rate of PKoV, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status in southern China, suggesting that controlling porcine diarrhea might be more complex than previously thought. A better understanding of viruses that cause diarrhea in piglets will aid in better preventing and controlling epidemics of viral porcine diarrhea.

scholarly journals Epidemiological analysis of Porcine Viral Diarrhea Pathogens in Local Area

2020 ◽  
Author(s):  
Ying Shi ◽  
Benqiang Li ◽  
Jie Tao ◽  
Jinghua Cheng ◽  
Huili liu
Keyword(s):  
Prevention And Control ◽  
Detection Method ◽  
Multiple Infection ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Multiplex Detection ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Pcr Method ◽  
And Control

Abstract Background Porcine viral diarrhea can cause great damage to the pig industry and high mortality to piglets. Furthermore, multiple pathogen infections and synergistic infections commonly existed in clinic. This has resulted in great difficulties in determining the main pathogenic factors, which would delay the prevention and control of diseases. Methods A total of 518 porcine stool specimens were collected from 9 pig herds in Shanghai, China from 2015 to 2017 and used for pathogen detection. A Luminex xTAG multiplex detection method was developed for the detection of 11 viral diarrhea pathogens, which allows for the simultaneous qualitative and quantitative detection of viral diarrhea pathogens in clinical samples. Results The minimum detection rate of the Luminex xTAG multiplex detection method was at least 10 times higher than the traditional PCR method. As a result, 209 (40.3%) were positive for porcine kobuvirus (PKoV), 138 (26.6%) for porcine astrovirus (PAstV), 91 (17.6%) for porcine epidemic diarrhea virus (PEDV), 78 (15.1%) for porcine sapelovirus (PSV), 68 (13.1%) for porcine sapovirus (PoSaV), 27 (5.2%) for porcine teschovirus (PTV), 25 (4.8%) for porcine deltacoronavirus (PDCoV), 17 (3.3%) for porcine rotavirus (PoRV), 16 (3.1%) for transmissible gastroenteritis virus (TGEV), 10 (1.9%) for porcine torovirus (PToV), and 9 (1.7%) for bovine viral diarrhea virus (BVDV), respectively. Furthermore, multiple infection rate of diarrhea sample was 17.57% for dual-infection, 11.58%for triple-infection, 4.63% for quadruple-infection, 0.77% for quintuple-infection, 0.58% for sextuple-infection and septuple-infection, respectively. Infection pattern of the viral diarrheal pathogens was changing, and different farm had the various diarrhea infection patterns, which proved the great importance of epidemiological surveillance and the guidance effect to clinical production. PoSaV, PoRV, PAstV, PToV and PEDV were indicated as the predominant viruses of clinical samples collected in 2017 by the quantitative analysis. Conclusions Here we provide a Luminex xTAG multiple detection method for viral diarrhea pathogen infection in clinical,which was more sensitive and specific than general multiplex PCR method. Furthermore, the surveillance confirmed the complicated infection status in China, which demonstrated the need for continuous surveillance and provided data for the prevention and control of viral diarrhea.

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.

Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy

2017 ◽  
Vol 63 (4) ◽  
pp. 296-302 ◽  
Author(s):  
Massimiliano Bergallo ◽  
Ilaria Galliano ◽  
Paola Montanari ◽  
Martina Rosa Brusin ◽  
Serena Finotti ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acute Gastroenteritis ◽  
Taqman Assay ◽  
Quantitative Detection ◽  
Common Disease ◽  
Median Viral Load ◽  
Significant Difference ◽  
Pcr Method ◽  
Children Under 5

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

scholarly journals A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid

2016 ◽  
Vol 10 (01) ◽  
pp. 53-61 ◽  
Author(s):  
Idrissa Diawara ◽  
Khalid Katfy ◽  
Khalid Zerouali ◽  
Houria Belabbes ◽  
Naima Elmdaghri
Keyword(s):  
Cerebrospinal Fluid ◽  
Bacterial Meningitis ◽  
Patient Management ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Pcr Method

Introduction: Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococcus pneumoniae in the cerebrospinal fluid (CSF) for meningitis diagnosis using SYBR Green-based RT-PCR method coupled with melting curve analysis. Methodology: We used SYBR Green-based RT-PCR method coupled with melting curve analysis to detect S. pneumoniae and N. meningitidis in CSF samples. The sensitivity, specificity, and limit of detection were determined. The gold standard for routine tests of CSF analysis is direct examination, culture, and/or latex agglutination. The assay was evaluated on 132 CSF samples to measure clinical sensitivity. Results: A duplex RT-PCR assay for N. meningitidis and S. pneumoniae detection in CSF was evaluated. Two peaks at different melting temperatures (87.5°C and 85.5°C) for N. meningitidis and S. pneumoniae, respectively, were obtained. The sensitivity of RT-PCR was 100% (95% confidence limits [CI] = 82.4–100) for N. meningitidis and 100% (95% CI = 85.1–100) for S. pneumoniae. Specificity was the same (100%) for the bacteria (95% CI = 88.6–100). The percentage of cases accurately diagnosed with meningitis caused by N. meningitidis and S. pneumoniae increased to 50.7% and 28.6%, respectively, when RT-PCR was added to the standard microbiologic methods. Conclusions: Duplex RT-PCR and melting curve analysis with SYBR Green is an inexpensive, sensitive, and specific method to rapidly diagnose bacterial meningitis. Accurate identification of the bacterial causative agents will improve patient management and epidemiological investigations.

scholarly journals Performance of Self-Collected Saliva Testing Compared with Nasopharyngeal Swab Testing for the Detection of SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 895
Author(s):  
Florence Carrouel ◽  
Martine Valette ◽  
Hervé Perrier ◽  
Maude Bouscambert-Duchamp ◽  
Claude Dussart ◽  
...  
Keyword(s):  
Viral Load ◽  
Quantitative Pcr ◽  
Statistical Analyses ◽  
Fair Agreement ◽  
Quantitative Detection ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Noninvasive Techniques ◽  
Concordance Probability ◽  
Salivary Test

The aim of this study was to determine whether self-collected pure saliva (SCPS) is comparable to nasopharyngeal (NP) swabs in the quantitative detection of SARS-CoV-2 by RT-PCR in asymptomatic, mild patients with confirmed COVID-19. Thirty-one patients aged from 18 to 85 years were included between 9 June and 11 December 2020. A SCPS sample and a NP sample were taken for each patient. Quantitative PCR was performed to detect SARS-CoV-2 viral load. Results of SCPS vs NP samples testing were compared. Statistical analyses were performed. Viral load was significantly correlated (r = 0.72). The concordance probability was estimated at 73.3%. In symptomatic adults, SCPS performance was similar to that of NP swabs (Percent Agreement = 74.1%; p = 0.11). Thus, the salivary test based on pure oral saliva samples easily obtained by noninvasive techniques has a fair agreement with the nasopharyngeal one in asymptomatic, mild patients with a confirmed diagnosis of COVID-19.

scholarly journals COVID-19 Biomarkers and Advanced Sensing Technologies for Point-of-Care (POC) Diagnosis

2021 ◽  
Vol 8 (7) ◽  
pp. 98
Author(s):  
Ernst Emmanuel Etienne ◽  
Bharath Babu Nunna ◽  
Niladri Talukder ◽  
Yudong Wang ◽  
Eon Soo Lee
Keyword(s):  
Response Times ◽  
Rna Virus ◽  
Point Of Care ◽  
Protein Detection ◽  
Turnaround Time ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Specificity And Sensitivity ◽  
Small Market ◽  
Multiple Biomarkers

COVID-19, also known as SARS-CoV-2 is a novel, respiratory virus currently plaguing humanity. Genetically, at its core, it is a single-strand positive-sense RNA virus. It is a beta-type Coronavirus and is distinct in its structure and binding mechanism compared to other types of coronaviruses. Testing for the virus remains a challenge due to the small market available for at-home detection. Currently, there are three main types of tests for biomarker detection: viral, antigen and antibody. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) remains the gold standard for viral testing. However, the lack of quantitative detection and turnaround time for results are drawbacks. This manuscript focuses on recent advances in COVID-19 detection that have lower limits of detection and faster response times than RT-PCR testing. The advancements in sensing platforms have amplified the detection levels and provided real-time results for SARS-CoV-2 spike protein detection with limits as low as 1 fg/mL in the Graphene Field Effect Transistor (FET) sensor. Additionally, using multiple biomarkers, detection levels can achieve a specificity and sensitivity level comparable to that of PCR testing. Proper biomarker selection coupled with nano sensing detection platforms are key in the widespread use of Point of Care (POC) diagnosis in COVID-19 detection.

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