Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples

Abstract Background: Early and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests. Here, we developed a RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. Results: Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction through the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The detection limit was ranging from 1×102 to 1×105 copies/ml for each diarrhea virus. Conclusions: In conclusion, the RRCMC method is a suitable rapid clinical test for infection viruses, with the advantages of high-throughput, low cost, high sensitivity, and specificity.

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Covid-19: A review of its clinical features, effects on gastrointestinal system and possibility of faecal transmission

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11ispl1.2881 ◽

2020 ◽

Vol 11 (SPL1) ◽

pp. 623-627

Author(s):

Kanishk K Adhit ◽

Anjankar Ashish P ◽

Siddhaarth K

Keyword(s):

Clinical Presentation ◽

Throat Swab ◽

Respiratory Illness ◽

Review Article ◽

Gastrointestinal System ◽

Rt Pcr ◽

Significant Evidence ◽

Stool Samples ◽

High Possibility ◽

Positive Results

In China, Wuhan in the province of China, COVID-19 a patient suffering from pneumonia was tested and to identify the cause, the throat swab of the patient was tested. On 7th January 2020 WHO declared the identification as COVID-19. And then it was proclaimed as a pandemic. It classically causes a respiratory illness presenting as a mild cough, fever and . However, several investigators have advocated the involvement of the gastrointestinal tract and liver in COVID-19 infection similar to other infections. Further research studies have shown results that are expanding the possibility of transmission because RT-PCR assessment has shown significant evidence for the presence of virus not only in samples but also in stool samples. Studies have shown that virus in stool samples have got positive results even after the illness has resolved, and two respiratory tests were done 24 hours after COVID-19 being tested negative. The review article the different findings of the clinical presentation of COVID-19. It sheds light on the effects of COVID-19 in the gastrointestinal system along with the reasons for the high possibility of transmission of COVID-19 through the route.

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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction

BioTechniques ◽

10.2144/05382rr05 ◽

2005 ◽

Vol 38 (2) ◽

pp. 287-293 ◽

Cited By ~ 183

Author(s):

Van Luu-The ◽

Nathalie Paquet ◽

Ezequiel Calvo ◽

Jean Cumps

Keyword(s):

Real Time ◽

High Throughput ◽

Second Derivative ◽

Rt Pcr ◽

Pcr Method

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Detection of norovirus in stool samples by RT-PCR in 5 disease centers in Iran

BMC Infectious Diseases ◽

10.1186/1471-2334-12-s1-o10 ◽

2012 ◽

Vol 12 (Suppl 1) ◽

pp. O10 ◽

Cited By ~ 1

Author(s):

Saadat Adabian ◽

Fatemeh Fallah ◽

Latif Gachkar ◽

Farzaneh Jadali ◽

S Tabatabaei ◽

...

Keyword(s):

Rt Pcr ◽

Stool Samples

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Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

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Electropherotypes and G-Types of Group A Rotaviruses Detected in Children with Diarrhea in Lagos, Nigeria

ISRN Virology ◽

10.5402/2013/179871 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 2

Author(s):

Christianah Idowu Ayolabi ◽

David Ajiboye Ojo ◽

George Enyimah Armah

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Migration Pattern ◽

Genomic Diversity ◽

Rt Pcr ◽

Group A Rotaviruses ◽

Group A ◽

Stool Samples ◽

Health Care Centers ◽

Virus Isolates

Approximately over 500,000 children die annually due to severe dehydrating diarrhea caused by rotaviruses. This work investigated rotavirus infection among children less than 5 years with diarrhea in Lagos and determined the circulating electropherotypes and genotypes of the virus isolates. Three hundred and two (n=302) stool samples from children below 60 months were collected from different hospitals and health care centers in Lagos and subjected to enzyme immunoassay (EIA) to determine the presence of Group A rotavirus, RT-PCR to determine the G-types, and polyacrylamide gel electrophoresis (PAGE) to determine the electropherotypes. The results show that 60.3% of the samples showed distinct rotavirus RNA migration pattern, having long electropherotypes (55.3%) of seven variations dominating over the short electropherotypes (44.5%). Six different G-types were detected (G1, G2, G3, G4, G9, and G12). Serotypes G1 and G12 showed long electropherotypic pattern while G2, G3, and G9 exhibited either short or long electropherotype. All G4 detected show short electropherotypic pattern. In conclusion, information on the genomic diversity and RNA electropherotypes of rotaviruses detected in children with diarrhea in Lagos is reported in this study.

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Expression profile of microRNAs in gastrointestinal stromal tumors revealed by high throughput quantitative RT-PCR microarray

World Journal of Gastroenterology ◽

10.3748/wjg.v21.i19.5843 ◽

2015 ◽

Vol 21 (19) ◽

pp. 5843-5855 ◽

Cited By ~ 13

Author(s):

Han-Xing Tong ◽

Yu-Hong Zhou ◽

Ying-Yong Hou ◽

Yong Zhang ◽

Yuan Huang ◽

...

Keyword(s):

Expression Profile ◽

High Throughput ◽

Gastrointestinal Stromal Tumors ◽

Rt Pcr ◽

Stromal Tumors ◽

Pcr Microarray

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High-throughput immunoassays for SARS-CoV-2, considerable differences in performance when comparing three methods

10.1101/2020.05.22.20106294 ◽

2020 ◽

Cited By ~ 1

Author(s):

Oskar Ekelund ◽

Kim Ekblom ◽

Sofia Somajo ◽

Johanna Pattison-Granberg ◽

Karl Olsson ◽

...

Keyword(s):

High Throughput ◽

Rapid Test ◽

Performance Criteria ◽

Clinical Use ◽

Rt Pcr ◽

Regulatory Authorities ◽

Flow Test ◽

Lateral Flow Test ◽

High Level ◽

Positive Results

Background: The recently launched high-throughput assays for the detection of antibodies against SARS-CoV-2 may change the managing strategies for the COVID-19 pandemic. This study aimed at investigating the performance of three high-throughput assays and one rapid lateral flow test relative to the recommended criteria defined by regulatory authorities. Methods: A total of 133 samples, including 100 pre-pandemic samples, 20 samples from SARS-CoV-2 RT-PCR positive individuals, and 13 potentially cross-reactive samples were analysed with SARS-CoV-2 IgG (Abbott), Elecsys Anti-SARS-CoV-2 (Roche), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) and 2019-nCOV IgG/IgM Rapid Test (Dynamiker Biotechnology Co). Results: All assays performed with a high level of specificity; however, only Abbott reached 100% (95% CI 96.3-100). The pre-pandemic samples analysed with Roche, DiaSorin and Dynamiker Biotechnology resulted in two to three false-positive results per method (specificity 96.9-98.0%). Sensitivity differed more between the assays, Roche exhibiting the highest sensitivity (100%, CI 83.9-100). The corresponding figures for Abbott, DiaSorin and Dynamiker Biotechnology were 85.0%, 77.8% and 75.0%, respectively. Conclusions: The results of the evaluated SARS-CoV-2 assays vary considerably as well as their ability to fulfil the performance criteria proposed by regulatory authorities. Introduction into clinical use in low-prevalent settings, should therefore, be made with caution.

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Immunological and molecular detection of rotavirus genotype in calves with gastroenteritis in Diyala-Iraq

10.53346/wjbpr.2021.1.1.0013 ◽

2021 ◽

Vol 1 (1) ◽

pp. 001-013

Author(s):

Ammar Talib Nasser ◽

Abdulrazak Shafiq Hasan ◽

Amer Khazaal Saleh ◽

Mohammad Kassem Saleh

Keyword(s):

Molecular Detection ◽

Cross Sectional Study ◽

Sectional Study ◽

Rt Pcr ◽

Cross Sectional ◽

Reverse Transcription Pcr ◽

Group A ◽

Stool Antigen ◽

Stool Samples ◽

Age Range

Aim: To explore the prevalence of rotavirus infection along with the molecular detection and genotyping of group A rotavirus (RVA) among bovine calves up to 5 months old in Diyala province-Iraq. Methods: This is a cross sectional study conducted in Diyala province-Iraq during the period of 2019-2020. One hundred bovine calves with age range of 1-5 months were included in the study. All were suffering acute gastroenteritis. Serum anti-rotavirus IgM and IgG plus fecal rotavirus Ag were tested for using ELISA techniques. Stool samples positive for rotavirus Ag were submitted for reverse transcription PCR (RT-PCR) for G and P genes, followed by sequencing and genotyping thereafter. Statistical analysis was done using SPSS version 25 and P values ≤ 0.05 were considered significant. Results: The positivity rate of anti-rotavirus IgM was 80% (P = 0.0001), and that of anti-rotavirus IgG was 79% (P = 0.0001). The rotavirus stool antigen was detected in 68% of calves (P = 0.01). A total of 45 stool samples which were positive for rotavirus Ag were submitted for RT-PCR; 13 (28.9%) were positive and 32 (71.1%) were negative (P = 0.084). 10 PCR positive samples were used for sequencing and genotyping and indicated that all investigated strains belonged to G1P[8] genotype. Conclusion: The current strains analyzed belonged to the G1P[8] RVA genotypes, affirming that employment of VP7 gene polymorphism accurately yielded uniform phylogenetic distances amongst investigated rotavirus strains and that there were no noticeable assortment events between human and animal rotavirus strains in Diyala province.

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