scholarly journals A Multiplex PCR Assay for Identifying All Major SARS-CoV-2 Variants

2021 ◽  
Author(s):  
Ryan J Dikdan ◽  
Salvatore A.E. Marras ◽  
Amanda P. Field ◽  
Alicia Brownlee ◽  
Alexander Cironi ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Deep Sequencing ◽  
Molecular Beacon ◽  
Limit Of Detection ◽  
Pcr Assay ◽  
Protein Coding ◽  
Qrt Pcr ◽  
Multiplex Pcr Assay ◽  
Testing Patient

Background Variants of Concern (VOC) of SARS-CoV-2, including the Alpha, Beta, Gamma, and Delta, threaten to prolong the pandemic leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the development and evolution of the virus, but is costly, slow, and not easily accessible. Methods A multiplex qRT-PCR assay for SARS-CoV-2 was developed, which identifies all VOC as well as other mutations of interest in the viral genome, eight mutations total, using single nucleotide discriminating molecular beacons in a two-tube assay. The sensitivity and specificity of the assay was tested using in vitro-transcribed targets. Twenty-six SARS-CoV-2 positive patient samples were blinded, then tested using this assay and compared with deep sequencing results. Findings The presented variant molecular beacon assay showed high accuracy when testing in vitro-transcribed targets, down to a limit of detection of five copies of the viral RNA, with 100% specificity. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26/26). Using this accurate genotyping, the SARS-CoV-2 samples were classified as the appropriate variants, and of the twenty-six samples two were identified as VOC Alpha, eight as VOC Delta, and two as Epsilon. Interpretation We have developed a qRT-PCR assay for the identification of currently circulating VOC of SARS-CoV-2 as well as other important mutations in its Spike protein coding sequence. This assay can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.

Molecular Identification of Alloreactive CTL Precursors in Hematopoietic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 597-597
Author(s):  
Christine O’Keefe ◽  
Lukasz Gondek ◽  
Ronald Sobecks ◽  
Alexander Rodriguez ◽  
Yadira Narvaez ◽  
...  
Keyword(s):  
T Cell ◽  
Quantitative Pcr ◽  
Direct Sequencing ◽  
Cell Receptor ◽  
Cytotoxic T Cell ◽  
Pcr Assay ◽  
Hematopoietic Stem ◽  
Multiplex Pcr Assay ◽  
Quantitative Pcr Assay

Abstract One of the leading complications in HSCT is GvHD mediated by allospecific cytotoxic T cell (CTL) clones that develop from pre-primed or naïve alloreactive precursors present in the graft. The ability to identify GvHD-specific CTL may facilitate development of monitoring tools for management of GvHD. The sequence (or clonotype) of the complementarity determining region 3 of the variable beta (VB) chain of T cell receptor (TCR) can be used as a molecular marker of individual T cell clones that can be detected in blood or tissues affected by GvHD. We have previously shown that although GvHD is a polyclonal process, immunodominant clonotypes and consequently CTL clones can be identified and quantitated (Exp. Hem.32: 1010–1022; Br. J. Haematol.129: 411–419). Although these clonotypes likely correspond to CTL clones participating in the GvHD process their specificity is unknown and may be directed against infectious agents or autoantigens. To more rationally utilize clonotypic diagnostics using truly allospecific CTL clones we developed new methods of selection of immunodominant alloreactive clonotypes. These clonotypes can be isolated from in vitro activated CTL populations following an allostimulation assay or from tissues affected by GvHD, indirectly indicating their target specificity. In 8 HSCT donor/patient pairs we performed allostimulation cocultures using recipient cells as targets and graft lymphocytes as effectors; activated donor CD8+ T cells were sorted based on CD69 expression and immunodominant CTL clonotypes were analyzed in the activated fraction. were studied. Several immunodominant clonotypes were identified and 11 were found to be restricted to activated CTL. The frequency of 5 immunodominant clonotypes from 2 patients was followed in the post-transplant course by direct sequencing of the appropriate VB family-specific PCR. For 4 of the 5 putatively allospecific clones a clonotype-specific quantitative PCR assay was designed and performed, illustrating that levels of the clonotypes in blood can be monitored using this technology. By direct sequencing, allospecific clonotypes were not detected in biopsies of GvHD-affected tissues. By more sensitive clonotype-specific quantitative PCR assay allostimulation-derived clonotype was also detected in skin biopsy obtained for GvHD diagnosis following transplantation. We have also used a reverse strategy for detection of allsopecific clonotypes. GvHD affected tissues were used as a source of clonotype isolation; VB-specific multiplex PCR assay performed on biopsy-derived DNA from 6/8 of the HSCT recipients was performed and a total of 12 immunodominant clonotypes were identified in 5 patients. We showed that identical CTL clones can be detected in various tissues affected by GvHD (e.g. gut and skin). Moreover, we demonstrate that tissue-derived clonotypes can also be used for clonotype-specific quantitative PCR. For example, 1/3 patients studied tissue-derived clonotype was detected also in blood. Our studies demonstrate the feasibility of identifying alloreactive precursors in HSC grafts pre-transplant or during the first bout of GvHD. Once such CTL marker clonotypes are identified, they may be selectively depleted from the graft or simply be used as an individualized marker of GvHD activity.

scholarly journals Evaluation of a Multiplex PCR Assay To Rapidly Detect Enterobacteriaceae with a Broad Range of β-Lactamases Directly from Perianal Swabs

2016 ◽  
Vol 60 (11) ◽  
pp. 6957-6961 ◽  
Author(s):  
Kalyan D. Chavda ◽  
Michael J. Satlin ◽  
Liang Chen ◽  
Claudia Manca ◽  
Stephen G. Jenkins ◽  
...  
Keyword(s):  
Hematopoietic Stem Cell Transplant ◽  
Molecular Beacon ◽  
Cell Transplant ◽  
Transplant Recipients ◽  
Pcr Assay ◽  
Hematopoietic Stem ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Highly Sensitive ◽  
Chromogenic Agar

ABSTRACTWe developed and evaluated multiplexed molecular beacon probes in a real-time PCR assay to identify prominent extended-spectrum-β-lactamase, plasmid-mediated AmpC β-lactamase (pAmpC) and carbapenemase genes directly from perianal swab specimens within 6 h. We evaluated this assay on 158 perianal swabs collected from hematopoietic stem cell transplant recipients and found that this assay was highly sensitive and specific for detection of CTX-M-, pAmpC-, and KPC-producingEnterobacteriaceaecompared to culture on chromogenic agar.

scholarly journals A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
Bladder Cancer ◽  
High Risk ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Allele Specific ◽  
Fgfr3 Mutations

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag ® , associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag ® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9 , HS3ST2 and SLIT2 methylated genes) assays. Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag ® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

2020 ◽  
Author(s):  
Boon-Teong Teoh ◽  
Kim-Ling Chin ◽  
Nur-Izyan Samsudin ◽  
Shih-Keng Loong ◽  
Sing-Sin Sam ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Zikv Infection ◽  
Qrt Pcr

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (k = 0.913, P < 0.001).Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

scholarly journals A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

2020 ◽  
Author(s):  
Boon-Teong Teoh ◽  
Kim-Ling Chin ◽  
Nur-Izyan Samsudin ◽  
Shih-Keng Loong ◽  
Sing-Sin Sam ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Zikv Infection ◽  
Qrt Pcr

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (k = 0.913, P < 0.001).Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

scholarly journals A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Boon-Teong Teoh ◽  
Kim-Ling Chin ◽  
Nur-Izyan Samsudin ◽  
Shih-Keng Loong ◽  
Sing-Sin Sam ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Zikv Infection ◽  
Qrt Pcr

Abstract Background Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required. Methods In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay. Results The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6–98.2) and 100% (95% CI = 78.5–100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001). Conclusion The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

scholarly journals Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Anny Armas Cayarga ◽  
Yenitse Perea Hernández ◽  
Yaimé J. González González ◽  
Santiago Dueñas Carrera ◽  
Idania González Pérez ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Rna Transcripts ◽  
Qrt Pcr ◽  
Immunodeficiency Virus ◽  
Limit Detection ◽  
Hiv 1

Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) byin vitrotranscription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy ( log10unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation () was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

scholarly journals A Multiplex PCR Assay for Detection of Pseudomonas syringae pv. actinidiae and Differentiation of Populations with Different Geographic Origin

Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 472-478 ◽  
Author(s):  
G. M. Balestra ◽  
M. C. Taratufolo ◽  
B. A. Vinatzer ◽  
A. Mazzaglia
Keyword(s):  
New Zealand ◽  
Pseudomonas Syringae ◽  
Limit Of Detection ◽  
Infected Plant ◽  
Geographic Origin ◽  
Detection Methods ◽  
Bacterial Canker ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Chinese Group

Pseudomonas syringae pv. actinidiae is responsible for severe outbreaks of bacterial canker of kiwifruit currently occurring around the world. Although molecular detection methods have been reported, none provide complete selectivity for this pathovar or discriminate among pathogen haplotypes. Therefore, a new multiplex polymerase chain reaction (PCR) assay was developed and validated. The assay was tested on 32 P. syringae pv. actinidiae isolates and 15 non-P. syringae pv. actinidiae strains and correctly assigned P. syringae pv. actinidiae strains to three different haplotypes: a Japanese/Korean group, a European group, and a Chinese group. Two P. syringae pv. actinidiae isolates from New Zealand were found to belong to the Chinese group whereas two other isolates from New Zealand, which were isolated from kiwifruit plants but which do not cause bacterial canker, tested negative. The described PCR assays has a limit of detection of approximately 5 to 50 pg of purified DNA or of 5 × 102 bacteria/PCR and were shown to work with both artificially and naturally infected plant tissues. Thus, the described method represents a suitable tool for detection of P. syringae pv. actinidiae and haplotype attribution, in particular, when testing a high number of samples during surveillance and prevention activities.

scholarly journals Development of New PCR Assay with SYBR Green I for Detection of Mycoplasma, Acholeplasma, and Ureaplasma sp. in Cell Cultures

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 876
Author(s):  
Jolanta Krzysztoń-Russjan ◽  
Jakub Chudziak ◽  
Małgorzata Bednarek ◽  
Elżbieta Lidia Anuszewska
Keyword(s):  
Cell Cultures ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Time Saving ◽  
Detection And Identification ◽  
Polymerase Chain

Mycoplasma, Acholeplasma, and Ureaplasma sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the Mycoplasma, Acholeplasma, and Ureaplasma sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of M. arginini, M. orale, M. hyorhinis, M. fermentans, M. genitalium, M. hominis, M. pneumoniae, M. salivarium, M. pirum, A. laidlawii, and U. urealyticum. Limit of detection values varied between 125–300 and 50–100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from Mycoplasma, Acholeplasma, and Ureaplasma in tested cell cultures.

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