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 Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

scholarly journals An enhanced isothermal amplification assay for viral detection

2020 ◽  
Author(s):  
Jason Qian ◽  
Sarah A. Boswell ◽  
Christopher Chidley ◽  
Zhi-xiang Lu ◽  
Mary E. Pettit ◽  
...  
Keyword(s):  
Rna Isolation ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Sample Collection ◽  
Amplification Method ◽  
Current State ◽  
Highly Sensitive ◽  
Amplification Assay ◽  
Molecular Diagnostic Test

AbstractRapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available.One sentence summarySensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.

Shortening distance of forward and reverse primers for nucleic acid isothermal amplification

2014 ◽  
Vol 395 (6) ◽  
pp. 679-684
Author(s):  
Qu Haitao ◽  
Zhang Wenchao ◽  
Zhang Xiaohui ◽  
Wang Xiujun ◽  
Li Sulong
Keyword(s):  
Nucleic Acid ◽  
Reaction Times ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Amplification Efficiency ◽  
Specific Method ◽  
Detection Techniques ◽  
Amplification Method ◽  
Reverse Primer ◽  
Short Time

Abstract Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60–65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40–60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template.

scholarly journals Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum

1970 ◽  
Vol 7 (2) ◽  
pp. 109-114 ◽  
Author(s):  
A Poudel ◽  
BD Pandey ◽  
B Lekhak ◽  
B Rijal ◽  
BR Sapkota ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Common Symptom ◽  
Bcg Vaccination ◽  
Loop Mediated Isothermal Amplification ◽  
16S Rna ◽  
Clinical Profiling

Background: Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. Objectives: The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Materials and methods: Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Result: Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (Χ2=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. Conclusion: As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal. Key words: clinical profiling; Sputum; DNA; LAMP; M. tuberculosis; Nepal DOI: 10.3126/kumj.v7i2.2701 Kathmandu University Medical Journal (2009) Vol.7, No.2 Issue 26, 109-114

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections

2013 ◽  
Vol 142 (1) ◽  
pp. 1-11 ◽  
Author(s):  
J. GRAY ◽  
L. J. COUPLAND
Keyword(s):  
Nucleic Acid ◽  
Simultaneous Detection ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Nucleic Acid Amplification Techniques ◽  
Infectious Gastroenteritis ◽  
The Usa ◽  
Acid Test

SUMMARYOn 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG®Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene – in-vitrodiagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

scholarly journals Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Zongyuan Chen ◽  
William R. Abrams ◽  
Eran Geva ◽  
Claudia J. de Dood ◽  
Jesús M. González ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Microfluidic Device ◽  
Modular Design ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Nucleic Acid Amplification ◽  
Antibody Detection ◽  
Confirmatory Test ◽  
Nucleic Acid Detection ◽  
Sample Processing

A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test.

scholarly journals Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1356
Author(s):  
Sangha Kwon ◽  
Ha Youn Shin
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Time Accuracy

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

scholarly journals Rapid and reliable distinguish of Morinda officinalis How by loop-mediated isothermal amplification (LAMP) assay

2020 ◽  
Author(s):  
Shuhan Wang ◽  
Aihua Sui ◽  
Yafei Han ◽  
Quan Zhou ◽  
Hang Xu ◽  
...  
Keyword(s):  
Low Cost ◽  
Lamp Assay ◽  
Its2 Sequence ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Nuclear Ribosomal Dna ◽  
Traditional Medicines ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Human Disorders

Abstract The medicinal plant Morinda officinalis How (MO), especially the root, has been frequently used in traditional medicines around the world as an herbal drug for treating variable human disorders and diseases. Various adulterations of MO were found for economic or production limitations. However, authentication of MO from its adulterants by LAMP has not yet been established. The present study introduces a commercially available nucleic acid amplification method, loop-mediated isothermal amplification (LAMP) assay for the distinguish of MO from its adulterants. In this method, we combined DNA barcodes technology to design 2 pairs independent LAMP primers, which based on the internal transcribed spacer 2 (ITS2) sequence of MO’s nuclear ribosomal DNA. Our results showed that the LAMP could amplify the samples as expected and successfully identify target MO, and the limit for DNA template preciseness was verified as 1 × 10− 1 pg/µl. All the visual or real-time turbidity detection was performed within 60 min at approximately 63 °C. The result showed that the LAMP assay and primers we designed have high accuracy and efficiency for the differentiation of MO and its adulterants. Our results illustrated that the proposed low-cost, fast and reliable LAMP assay without the need for expensive equipment or specialized techniques could be a good way for MO rapid authentication.

Comparison of Diagnostic Yield of Tuberculosis Loop-Mediated Isothermal Amplification Assay With Cartridge-Based Nucleic Acid Amplification Test, Acid-Fast Bacilli Microscopy, and Mycobacteria Growth Indicator Tube Culture in Children With Pulmonary Tuberculosis

2020 ◽  
Author(s):  
Rajeshwar Dayal ◽  
Alok Yadav ◽  
Dipti Agarwal ◽  
Manoj Kumar ◽  
Raj Kamal ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Yield ◽  
Lamp Assay ◽  
Nucleic Acid Amplification Test ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Pulmonary Tb ◽  
Indicator Tube ◽  
Mgit Culture ◽  
Growth Indicator

Abstract Background Cartridge-based nucleic acid amplification test (CB-NAAT) has been recommended for diagnosis of tuberculosis (TB) in children, but its wide use is limited by high cost and the need for well-equipped laboratories. This study was conducted in children with pulmonary TB to compare the diagnostic yield of TB-LAMP (loop-mediated isothermal amplification test) with CB-NAAT and other conventional methods. Methods Patients ≤ 14 years of age diagnosed with probable pulmonary TB were included in the study. Induced sputum/gastric aspirate was obtained and subjected to acid-fast bacilli (AFB) microscopy, mycobacteria growth indicator tube (MGIT) culture, CB-NAAT, and TB-LAMP. The TB-LAMP assay was performed using 2 different primers, IS6110 and mpb64, for detection of Mycobacterium tuberculosis (MTB). TB-LAMP assays were compared to other assays using appropriate statistical tests. Results One hundred fourteen subjects were recruited in the study. AFB microscopy, MGIT culture, CB-NAAT, TB-LAMP IS6110, and TB-LAMP mpb64 showed positivity of 32 (28.1%), 59 (51.7%), 66 (57.9%), 75 (65.8%), and 81 (71%), respectively. TB-LAMP IS6110 showed significantly higher MTB detection in comparison to AFB microscopy and MGIT culture (P = .0001 and P = .03, respectively), and showed no significant difference in MTB detection in comparison with CB-NAAT (P = .219). TB-LAMP mpb64 showed significantly higher MTB detection as compared to AFB microscopy, MGIT culture, and CB-NAAT (P = .0001, P = .003, and P = .037, respectively). TB-LAMP mpb64 and IS6110 showed sensitivity of 94.9% (95% confidence interval [CI], 85.9%–98.9%) and 89.8% (95% CI, 79.7%–96.2%), respectively, in reference to MGIT culture. The degree of agreement between TB-LAMP (mpb64 and IS6110) with CB-NAAT showed κ values of 0.718 and 0.834, respectively. Conclusions TB-LAMP assay can be a useful alternative test in diagnosis of pulmonary TB in children.

scholarly journals Development and comparison of novel multiple cross displacement amplification (MCDA) assays with other nucleic acid amplification methods for SARS-CoV-2 detection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Laurence Don Wai Luu ◽  
Michael Payne ◽  
Xiaomei Zhang ◽  
Lijuan Luo ◽  
Ruiting Lan
Keyword(s):  
Nucleic Acid ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Rt Pcr ◽  
Amplification Method ◽  
Multiple Cross ◽  
Time To Detection

AbstractThe development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.

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