scholarly journals Identification of bacteria strains using the Recombinase Polymerase Amplification assay on a miniaturized solid-state pH sensor

2022 ◽  
Author(s):  
Anh H. Nguyen ◽  
Samir Malhotra ◽  
Michael P.H. Lau ◽  
Hung Cao
Keyword(s):  
Solid State ◽  
Nucleic Acid ◽  
Ph Sensor ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification ◽  
Optical Systems ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
E Coli ◽  
Identification Of Bacteria

Rapid identification of bacteria based on nucleic acid amplification allows dealing with the detection of pathogens in clinical, food, and environmental samples. Amplification products must be detected and analyzed by external devices or integrated complicated optical systems. Here, we developed a solid-state pH electrode based on iridium oxide (IrO2) films to measure released hydrogen ions (H+) from isothermal nucleic acid (NA) amplification of bacterial samples. By recombinase polymerase amplification (RPA), we achieved rapid (< 15 min) and sensitive (<30 copies) detection with an accuracy of about 0.03 pH. The RPA-based hydrogen ion sensing assay shows higher specificity, sensitivity, and efficiency as the same polymerase chain reaction (PCR) methods. We initially used the RPA-based sensor to detect E. coli species in laboratory samples. Among, 27 random laboratory samples of E. coli samples, 6 were found to be DH5alpha, 9 BL21, 3 HB101, 6 TOP10, and 3 JM109. The electrical detection of amplification provides generally applicable techniques for the detection of nucleic acid amplification, enabling molecular diagnostic tests in the field and integrating data transmission to the mobile device. These results can be future developed into an efficient tool for rapid on-site detection of bacterial pathogens in clinical samples.

scholarly journals Comparative performance and cycle threshold values of 10 nucleic acid amplification tests for SARS-CoV-2 on clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252757
Author(s):  
Miyuki Mizoguchi ◽  
Sohei Harada ◽  
Koh Okamoto ◽  
Yoshimi Higurashi ◽  
Mahoko Ikeda ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Performance ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Nucleic Acid Amplification Tests ◽  
Cycle Threshold ◽  
E Gene ◽  
Viral Copy Number ◽  
Gene Test ◽  
Positive Results

Background A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. Methods We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. Results Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/μL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/μL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. Conclusion Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2

2021 ◽  
Vol 11 ◽  
Author(s):  
Pei Wang ◽  
Chao Ma ◽  
Xue Zhang ◽  
Lizhan Chen ◽  
Longyu Yi ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rapid Detection ◽  
Simple Procedure ◽  
Quantitative Polymerase Chain Reaction ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
High Concentration ◽  
Sample Processing ◽  
Low Efficiency

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

scholarly journals Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in yaws-like lesions

2020 ◽  
Author(s):  
Michael Frimpong ◽  
Shirley Victoria Simpson ◽  
Hubert Senanu Ahor ◽  
Abigail Agbanyo ◽  
Solomon Gyabaah ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Treponema Pallidum ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification ◽  
Haemophilus Ducreyi ◽  
World Health ◽  
Clinical Samples ◽  
Successful Implementation ◽  
Effective Diagnosis

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a duplex RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). TPHD-RPA assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 minutes at 42 oC. The duplex RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Compared with commercial multiplex real-time PCR, the TPHD-RPA assay demonstrated 94-95% sensitivity for T. pallidum and H. ducreyi confirmed samples, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication programs by ensuring effective diagnosis as well as enable monitoring of eradication efforts success or failure and planning of follow-up interventions at the community level.

scholarly journals Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection

2021 ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Study ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Predictive Values ◽  
Health Crisis ◽  
Sensitivity Specificity

Abstract Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic. Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)

scholarly journals Evaluation of AMPLILINK Software for the COBAS AMPLICOR System

1999 ◽  
Vol 37 (2) ◽  
pp. 436-437 ◽  
Author(s):  
Harald H. Kessler ◽  
Donald Jungkind ◽  
Evelyn Stelzl ◽  
Sue Direnzo ◽  
Srinivas K. Vellimedu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Diagnostic Procedures ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Single Specimen ◽  
Clinical Laboratories ◽  
And Control

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.

1997 ◽  
Vol 10 (2) ◽  
pp. 242-256 ◽  
Author(s):  
M Ieven ◽  
H Goossens
Keyword(s):  
Nucleic Acid ◽  
Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Clinical Laboratory ◽  
Pneumocystis Carinii ◽  
Diagnostic Techniques ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Amplification Techniques ◽  
Tract Infections

Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for excluding false-positive and false-negative results.

HIV Detection from Human Serum with Paper-Based Isotachophoretic RNA Extraction and Reverse Transcription Recombinase Polymerase Amplification

2021 ◽  
Author(s):  
Andrew Bender ◽  
Benjamin Sullivan ◽  
Jane Zhang ◽  
David Juergens ◽  
Lorraine Lillis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Human Serum ◽  
Internal Control ◽  
Reverse Transcription ◽  
Low Cost ◽  
Rna Extraction ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification ◽  
People Living With Hiv ◽  
Ms2 Bacteriophage

<p>The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy. These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5,000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.</p>

Different Methods for Molecular and Rapid Detection of Human Novel Coronavirus

2021 ◽  
Vol 27 ◽  
Author(s):  
Mohamad Hesam Shahrajabian ◽  
Wenli Sun ◽  
Qi Cheng
Keyword(s):  
Nucleic Acid ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Scientific Information ◽  
Meat Products ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
Nucleic Acid Amplification

Introduction: While PCR has been recognized as one of the appropriate ways to diagnosis of infectious diseases, Loop-mediated isothermal amplification (LAMP), which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords were PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA has benefited from isothermal PCR and both simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and employs at a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.

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