Optimizing SARS-CoV-2 Molecular Diagnostic Using N Gene Target

2020 ◽  
Author(s):  
Raphael Contelli Klein ◽  
Mary Hellen Fabres-Klein ◽  
Larissa Gomes Barbosa ◽  
Lívia Vasconcelos Gonzaga Knnup ◽  
Larissa Paola Rodrigues Venâncio ◽  
...  
Keyword(s):  
Molecular Diagnostic ◽  
N Gene ◽  
Gene Target

scholarly journals Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Jonathan Hon-Kwan Chen ◽  
Cyril Chik-Yan Yip ◽  
Jasper Fuk-Woo Chan ◽  
Rosana Wing-Shan Poon ◽  
Kelvin Kai-Wang To ◽  
...  
Keyword(s):  
Hong Kong ◽  
High Throughput ◽  
Clinical Performance ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Gene Target ◽  
E Gene

ABSTRACT In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.

scholarly journals Optimizing SARS-CoV-2 molecular diagnostic using N gene target: insights about reinfection

2020 ◽  
Author(s):  
Raphael Contelli Klein ◽  
Mary Hellen Fabres Klein ◽  
Larissa Gomes Barbosa ◽  
Lívia Vasconcelos Gonzaga Knnup ◽  
Larissa Paola Rodrigues Venâncio ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Molecular Test ◽  
Gene Target ◽  
Infected People ◽  
Remote Locations ◽  
The World ◽  
The Cost ◽  
Huge Challenge

AbstractIntroductionMolecular diagnosis of SARS-CoV-2 is a huge challenge to many countries around the world. The cost of tests to check infected people is inaccessible since specialized teams and equipment are not disposable in remote locations. Herein, we compared the fitness of two primers sets to the SARS-CoV-2 N gene in the molecular diagnosis of COVID-19.Materials and MethodsThe 1029 patient samples were tested to presense/abscence molecular test using in house US CDC protocol. We compared the fitness of two primers sets to two different regions of N gene targets.ResultsBoth targets, N1 and N2 displayed similar fitness during testing with no differences between Ct or measurable viral genome copies. In addition, we verified security ranges Cts related to positive diagnostic with Ct above 35 value failuring in 66,6% after retesting of samples.Main conclusionOur data suggest that it is secure to use just one primer set to the N gene to identify SARS-CoV-2 in samples and the labs should be careful to set positive samples in high Ct values using high cutoffs.

scholarly journals Comparison of genes as target for molecular diagnosis of peste des petits ruminants in goats

2013 ◽  
Vol 29 (2) ◽  
pp. 56-62 ◽  
Author(s):  
AR Bhuiyan ◽  
MM Rahman ◽  
JA Begum ◽  
MR Islam ◽  
EH Chowdhury
Keyword(s):  
Viral Disease ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Peste Des Petits Ruminants ◽  
Gene Target ◽  
Field Samples ◽  
Successful Control ◽  
Primer Sets ◽  
Molecular Diagnostic Test ◽  
Np Gene

Peste des petits ruminants (PPR) are an acute viral disease of sheep and goats. Rapid and accurate diagnosis is essential for successful control. Reverse transcriptase polymerase chain reaction (RT-PCR), a molecular diagnostic test based on amplification of the gene target is more sensitive than other tests. The study was to find an efficient primer set and structural gene, which would be more specific and sensitive for detecting PPR virus (PPRV) in field samples. Six primer sets for six structural genes of PPR were used. Primer against NP gene (np3/np4) was specific and sensitive. To ensure efficient amplification and detection of viruses in field samples, more than one set of primers should be used and F and N gene specific primers were the most suitable. DOI: http://dx.doi.org/10.3329/bvet.v29i2.14343 Bangl. vet. 2012. Vol. 29, No. 2, 56-62

scholarly journals Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
S. Battaglia ◽  
A. Spitaleri ◽  
A. M. Cabibbe ◽  
C. J. Meehan ◽  
C. Utpatel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genetic Variants ◽  
Protein Structures ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Clear Understanding ◽  
Data Set ◽  
Gene Target ◽  
Genomic Regions

ABSTRACT The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants’ role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

scholarly journals An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

2022 ◽  
Author(s):  
Bongkot Ngamsom ◽  
Alexander Iles ◽  
Moses Kamita ◽  
Racheal Kimani ◽  
Pablo Rodriguez-Mateos ◽  
...  
Keyword(s):  
Saliva Sample ◽  
Rna Extraction ◽  
Lab On A Chip ◽  
Cross Reactivity ◽  
Contact Tracing ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Sample Collection ◽  
Middle Income ◽  
Specificity And Sensitivity

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

scholarly journals Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

2021 ◽  
Author(s):  
Pedro Augusto Alves ◽  
Ellen G. Oliveira ◽  
Ana Paula M. Franco-Luiz ◽  
Leticia T. Almeida ◽  
Amanda B. Goncalves ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
High Specificity ◽  
Cross Reactivity ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Clinical Validation ◽  
Rna Detection ◽  
Viral Transport Medium

The COVID-19 pandemics unfolded due to the widespread SARS-CoV-2 transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric RT-LAMP (reverse transcription loop-mediated isothermal amplification) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 viral genomic copies/uL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65 C for 30 min. When compared to RT-qPCR, up to Ct value 32, RT-LAMP presented 97% (87.4-99.4% 95% CI) sensitivity and 100% (86.2-100%) specificity for SARS-CoV-2 RNA detection targeting N gene. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI), such as variants occurring in Brazil named P.1, P.2, B.1.1.374 and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipments, infrastructure and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

scholarly journals COVID-19 cases from the first local outbreak of SARS-CoV-2 B.1.1.7 variant in China presented more serious clinical features: a prospective, comparative cohort study

2021 ◽  
Author(s):  
Yang Song ◽  
Ziruo Ge ◽  
Shuping Cui ◽  
Di Tian ◽  
Gang Wan ◽  
...  
Keyword(s):  
Cohort Study ◽  
Clinical Features ◽  
Ground Glass Opacity ◽  
N Gene ◽  
Gene Target ◽  
Clinical Indicators ◽  
Reactive Protein ◽  
Amyloid A ◽  
Significant Difference ◽  
Amino Acid Mutations

Background: The SARS-CoV-2 B.1.1.7 variant which was first identified in the United Kingdom (U.K.) has increased sharply in numbers worldwide and was reported to be more contagious. On January 17, 2021, a COVID-19 clustered outbreak caused by B.1.1.7 variant occurred in a community in Daxing District, Beijing, China. Three weeks prior, another non-variant (lineage B.1.470) COVID-19 outbreak occurred in Shunyi District, Beijing. This study aimed to investigate the clinical features of B.1.1.7 variant infection. Methods: A prospective cohort study was conducted on COVID-19 cases admitted to Ditan hospital since January 2020. Data of 74 COVID-19 cases from two independent COVID-19 outbreaks in Beijing were extracted as study subjects from a Cloud Database established in Ditan hospital, which included 41 Shunyi cases (Shunyi B.1.470 group) and 33 Daxing cases (Daxing B.1.1.7 group) that have been hospitalized since December 25, 2020 and January 17, 2021, respectively. We conducted a comparison of the clinical characteristics, RT-qPCR results and genomic features between the two groups. Findings: Cases from Daxing B.1.1.7 group (15 [45.5%] male; median age, 39 years [range, 30.5, 62.5]) and cases from Shunyi B.1.470 group (25 [61.0%] male; median age, 31 years [range, 27.5, 41.0]) had a statistically significant difference in median age (P =0.014). Seven clinical indicators of Daxing B.1.1.7 group were significantly higher than Shunyi B.1.470 group including patients having fever over 38℃ (14/33 [46.43%] in Daxing B.1.1.7 group vs. 9/41 (21.95%) in Shunyi B.1.470 group [P = 0 .015]), C-reactive protein ([CRP, mg/L], 4.30 [2.45, 12.1] vs. 1.80, [0.85, 4.95], [P = 0.005]), Serum amyloid A ([SAA, mg/L], 21.50 [12.50, 50.70] vs. 12.00 [5.20, 26.95], [P = 0.003]), Creatine Kinase ([CK, U/L]), 110.50 [53.15,152.40] vs. 70.40 [54.35,103.05], [P = 0.040]), D-dimer ([DD, mg/L], 0.31 [0.20, 0.48] vs. 0.24 [0.17,0.31], [P = 0.038]), CD4+ T lymphocyte ([CD4+ T, mg/L], [P = 0.003]) , and Ground-glass opacity (GGO) in lung (15/33 [45.45%] vs. 5/41 [12.20%], [P =0.001]). After adjusting for the age factor, B.1.1.7 variant infection was the risk factor for CRP (P = 0.045, Odds ratio [OR] 2.791, CI [1.025, 0.8610]), SAA (0.011, 5.031, [1.459, 17.354]), CK (0.034, 4.34, [0.05, 0.91]), CD4+ T ( 0.029, 3.31, [1.13, 9.71]), and GGO (0.005, 5.418, [1.656, 17.729]) of patients. The median Ct value of RT-qPCR tests of the N-gene target in the Daxing B.1.1.7 group was significantly lower than the Shunyi B.1.470 group (P=0.036). The phylogenetic analysis showed that only 2 amino acid mutations in spike protein were detected in B.1.470 strains while B.1.1.7 strains had 3 deletions and 7 mutations. Interpretation: Clinical features including a more serious inflammatory response, pneumonia and a possible higher viral load were detected in the cases infected with B.1.1.7 SARS-CoV-2 variant. It could therefore be inferred that the B.1.1.7 variant may have increased pathogenicity.

scholarly journals SARS-CoV-2 detection in multi-sample pools in a real pandemic scenario: a screening strategy of choice for active surveillance

2021 ◽  
Author(s):  
Andr&eacutes Marcos Castellaro ◽  
Pablo Velez ◽  
Guillermo Giaj Merlera ◽  
Juan Rondan Duenas ◽  
Felix Condat ◽  
...  
Keyword(s):  
Active Surveillance ◽  
Cost Benefit ◽  
Screening Strategy ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Acid Extraction ◽  
Virus Propagation ◽  
Gold Standard Method ◽  
Value Shift ◽  
Propagation Methods

Background The current COVID-19 pandemic has overloaded the diagnostic capacity of laboratories by the gold standard method rRT-PCR. This disease has a high spread rate and almost a quarter of infected individuals never develop symptoms. In this scenario, active surveillance is crucial to stop the virus propagation. Methods Between July 2020 and April 2021, 11580 oropharyngeal swab samples collected in closed and semi-closed institutions were processed for SARS-CoV-2 detection in pools, implementing this strategy for the first time in Cordoba, Argentina. Five-sample pools were constituted before nucleic acid extraction and amplification by rRT-PCR. Comparative analysis of cycle threshold (Ct) values from positive pools and individual samples along with a cost-benefit report of the whole performance of the results was performed. Results From 2314 5-sample pools tested, 158 were classified as positive (6.8%), 2024 as negative (87.5%), and 132 were categorized as indeterminate (5.7%). The Ct value shift due to sample dilution showed an increase in Ct of 2.6 ±1.53 cycles for N gene and 2.6 ±1.78 for ORF1ab gene. Overall, 290 pools were disassembled and 1450 swabs were analyzed individually. This strategy allowed correctly identifying 99.8% of the samples as positive (7.6%) or negative (92.2%), avoiding the execution of 7,806 rRT-PCR reactions which represents a cost saving of 67.5%. Conclusion This study demonstrates the feasibility of pooling samples to increase the number of tests performed, helping to maximize molecular diagnostic resources and reducing the work overload of specialized personnel during active surveillance of the COVID-19 pandemic.

scholarly journals Localised community circulation of SARS-CoV-2 viruses with an increased accumulation of single nucleotide polymorphisms that adversely affect the sensitivity of real-time reverse transcription assays targeting Nucleocapsid protein

2021 ◽  
Author(s):  
Catherine Moore ◽  
Louise Davies ◽  
Rhiannydd Rees ◽  
Laura Gifford ◽  
Heather Lewis ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Quality Management System ◽  
Data Availability ◽  
N Gene ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Gene Target ◽  
Additional Mutation ◽  
Target Sites ◽  
The Impact

SummaryCurrently the primary method for confirming acute SARS-CoV-2 infection is through the use of molecular assays that target highly conserved regions within the viral genome. Many, if not most of the diagnostic targets currently in use were produced early in the pandemic, using genomes sequenced and shared in early 2020. As viral diversity increases, mutations may arise in diagnostic target sites that have an impact on the performance of diagnostic tests. Here, we report on a local outbreak of SARS-CoV-2 which had gained an additional mutation at position 28890 of the nucleocapsid protein, on a background of pre-existing mutations at positions 28881, 28882, 28883 in one of the main circulating viral lineages in Wales at that time. The impact of this additional mutation had a statistically significant impact on the Ct value reported for the N gene target designed by the Chinese CDC and used in a number of commercial diagnostic products. Further investigation identified that, in viral genomes sequenced from Wales over the summer of 2020, the N gene had a higher rate of mutations in diagnostic target sites than other targets, with 115 issues identified affecting over 10% of all cases sequenced between February and the end of August 2020. In comparison an issue was identified for ORFab, the next most affected target, in less than 1.4% of cases over the same time period. This work emphasises the potential impact that mutations in diagnostic target sites can have on tracking local outbreaks, as well as demonstrating the value of genomics as a routine tool for identifying and explaining potential diagnostic primer issues as part of a laboratory quality management system. This work also indicates that with increasing genomic sequencing data availability, there is a need to re-evaluate the diagnostic targets that are in use for SARS-CoV-2 testing, to better target regions that are now demonstrated to be of lower variability.

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