scholarly journals Two target genes based multiple cross displacement amplification combined with a lateral flow biosensor for the detection of Mycobacterium tuberculosis complex

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Junfei Huang ◽  
Ziyu Xiao ◽  
Xinggui Yang ◽  
Xu Chen ◽  
Xiaojuan Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Genomic Dna ◽  
Target Genes ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Product Analysis ◽  
Prevention And Treatment ◽  
Multiple Cross ◽  
Colorimetric Indicator

Abstract Background Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). Hence, the development of a novel, simple, rapid and sensitive method to detect MTBC is of great significance for the prevention and treatment of TB. Results In this study, multiple cross displacement amplification (MCDA) combined with a nanoparticle-based lateral flow biosensor (LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC (MCDA-LFB). One suite of specific MCDA primers designed for the IS6110 and mpb64 genes was validated using genomic DNA extracted from the reference strain H37Rv. The MCDA amplicons were analyzed using a real-time turbidimeter, colorimetric indicator (malachite green, MG) and LFBs. The optimal amplification temperature and time were confirmed, and the MCDA-LFB method established in the current report was evaluated by detecting various pathogens (i.e., reference strains, isolates and clinical sputum samples). The results showed that the two sets of MCDA primers targeting the IS6110 and mpb64 genes could effectively detect MTBC strains. The optimal reaction conditions for the MCDA assay were determined to be 67 °C for 35 min. The MCDA assay limit of detection (LoD) was 100 fg per reaction for pure genomic DNA. The specificity of the MCDA-LFB assay was 100%, and there were no cross-reactions for non-MTBC strains. For sputum samples and MTBC strain detection, the positive rate of MCDA-LFB for the detection of MTBC strains was consistent with seminested automatic real-time PCR (Xpert MTB/RIF) and higher than acid-fast staining (AFS) and culture assays when used for sputum samples. The MCDA-LFB assay was a rapid tool, and the whole procedure for MCDA-LFB, including DNA template preparation, MCDA reaction and amplification product analysis, was completed within 70 min. Conclusion The MCDA-LFB assay targeting the IS6110 and mpb64 genes is a simple, rapid, sensitive and reliable detection method, and it has potential significance for the prevention and treatment of TB.

scholarly journals Two Target Genes Based Multiple Cross Displacement Amplification Combined with Lateral Flow Biosensor for the Detection of Mycobacrterium Tuberculosis Complex

2020 ◽  
Author(s):  
Junfei Huang ◽  
Ziyu Xiao ◽  
Xinggui Yang ◽  
Xu Chen ◽  
Xiaojuan Wang ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Genomic Dna ◽  
Target Genes ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Prevention And Treatment ◽  
Multiple Cross ◽  
Positive Rate

Abstract Background: Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB), which is a global public health problem that seriously endangers public health. Hence, development of a new and rapid method to detect MTBC is of great significance for prevention and treatment of TB. Results: In this study, a multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC. One suit of specific multiple cross displacement amplification (MCDA) primers, which was designed for IS6110 and mpb64 gene, respectively, was validated through using the genomic DNA extracted from reference strain H37Rv. The MCDA products were analyzed using real-time turbidity curve, colorimetric indicator (Malachite Green, MG) and LFB. The conditions of amplification temperature and time were optimized and the established MCDA-LFB method was applied to detect the sputum specimens and MTBC strains from clinical samples. The results show that two sets of MCDA primers for the IS6110 and mpb64 genes have detected MTBC validly. The MCDA reaction conditions were optimized at 67 °C for 35 min. The limit of detection of MCDA assay based on IS6110 and mpb64 genes was 100 fg of genomic DNA template per reaction. The specificity of MCDA-LFB detection was 100%, and no cross-reactions for non-MTBC strains detection. The positive rate of MCDA-LFB for the detection of MTBC strains was equal to that of semi-nested automatic real-time PCR (Xpert MTB/RIF), and had a higher positive rate than acid-fast staining (AFS) when used for the detection of sputum samples. The whole procedure of MCDA-LFB, including genomic template preparation, MCDA reaction and products analysis, was completed with 70 min.Conclusion: The simplicity, rapidity, sensitivity and reliability of the MCDA-LFB based on IS6110 and mpb64 gene of MTBC developed in this study make it potentially significant for the prevention and treatment of TB.

scholarly journals Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

2007 ◽  
Vol 73 (20) ◽  
pp. 6557-6565 ◽  
Author(s):  
Pascal E. Saikaly ◽  
Morton A. Barlaz ◽  
Francis L. de los Reyes
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Fate And Transport ◽  
Biological Warfare ◽  
Quantitative Real Time Pcr ◽  
Pcr Assays ◽  
Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

scholarly journals Identification of Acinetobacter baumannii and its carbapenem-resistant gene blaOXA-23-like by multiple cross displacement amplification combined with lateral flow biosensor

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shoukui Hu ◽  
Lina Niu ◽  
Fan Zhao ◽  
Linlin Yan ◽  
Jinqing Nong ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Conventional Culture ◽  
Carbapenem Resistant ◽  
Multiple Cross ◽  
Pure Cultures ◽  
Amplification Technique ◽  
Sensitivity Specificity

AbstractAcinetobacter baumannii is a frequent cause of the nosocomial infections. Herein, a novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene. The duplex MCDA assay, which targets the pgaD and blaOXA-23-like genes, could identify the A. baumannii isolates and differentiate these isolates harboring blaOXA-23-like gene. The disposable lateral flow biosensors (LFB) were used for analyzing the MCDA products. A total of sixty-eight isolates, include fifty-three A. baumannii strains and fifteen non-A. baumannii strains, were employed to optimize MCDA methods and determine the sensitivity, specificity and feasibility. The optimal reaction condition is found to be 63 °C within 1 h, with limit of detection at 100 fg templates per tube for pgaD and blaOXA-23-like genes in pure cultures. The specificity of this assay is 100%. Moreover, the practical application of the duplex MCDA-LFB assay was evaluated using clinical samples, and the results obtained from duplex MCDA-LFB method were consistent with conventional culture-based technique. In sum, the duplex MCDA-LFB assay appears to be a reliable, rapid and specific technique to detect all A. baumannii strains and identify these strains harboring blaOXA-23-like gene for appropriate antibiotic therapy.

scholarly journals Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Xin-na Li ◽  
Xin-xin Shen ◽  
Ming-hui Li ◽  
Ju-ju Qi ◽  
Rui-huan Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Enterovirus 71 ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Coxsackievirus A16 ◽  
Fluorescence Detector ◽  
Lateral Flow Strip ◽  
Clinical Diagnostic

Abstract Background Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Methods Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. Results The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. Conclusions The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

scholarly journals Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinggui Yang ◽  
Junfei Huang ◽  
Xu Chen ◽  
Ziyu Xiao ◽  
Xiaojuan Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Clinical Specimen ◽  
Lamp Assay ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Whole Process ◽  
Specimen Processing ◽  
Mycobacterium Africanum

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66°C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 × 103 CFU/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1–2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.

scholarly journals Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

2020 ◽  
Author(s):  
Jinfeng Wang ◽  
Ruiwen Li ◽  
Xiaoxia Sun ◽  
Libing Liu ◽  
Xuepiao Hao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Metal Bath ◽  
Lateral Flow Strip ◽  
The Real ◽  
Mycoplasmal Pneumonia ◽  
Mycoplasma Ovipneumoniae

Abstract Background: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminant s . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100% and 98.73%, diagnostic sensitivity of 90.63% and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. Conclusions: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

scholarly journals Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Luxi Jiang ◽  
Rumeng Gu ◽  
Xiaomeng Li ◽  
Meijun Song ◽  
Xiaojun Huang ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Culture Method ◽  
Lateral Flow ◽  
Nucleic Acid Amplification ◽  
Optimal Time ◽  
Target Sequence ◽  
Multiple Cross ◽  
Pure Cultures

Abstract Background Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires’ disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila. Results A set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher. Conclusions In summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.

scholarly journals Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays

2004 ◽  
Vol 70 (7) ◽  
pp. 4303-4317 ◽  
Author(s):  
Sung-Keun Rhee ◽  
Xueduan Liu ◽  
Liyou Wu ◽  
Song C. Chong ◽  
Xiufeng Wan ◽  
...  
Keyword(s):  
Microbial Community ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Target Genes ◽  
Metal Resistance ◽  
Detection Sensitivity ◽  
Pcr Analysis ◽  
Polyaromatic Hydrocarbon ◽  
Soil Microcosms

ABSTRACT To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50°C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was ∼5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 × 107 cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r2 = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r2 = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.

scholarly journals Rapid Monitoring of Vancomycin Concentration in Serum Using Europium (III) Chelate Nanoparticle-Based Lateral Flow Immunoassay

2021 ◽  
Vol 9 ◽  
Author(s):  
Lun Bian ◽  
Junyu Liang ◽  
Hui Zhao ◽  
Ke Ye ◽  
Zhaoyue Li ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Real Time Monitoring ◽  
Competitive Immunoassay ◽  
Vancomycin Concentration ◽  
The Real ◽  
Coefficients Of Variation ◽  
Rapid Monitoring

Establishing personalized medication plans for patients to maximize therapeutic efficacy and minimize the toxicity of vancomycin (VAN) requires rapid, simple, and accurate monitoring of VAN concentration in body fluid. In this study, we have developed a simple and rapid analytical method by integrating Eu (III) chelate nanoparticles (CN-EUs) and lateral flow immunoassay (LFIA) to achieve the real-time monitoring of VAN concentration in serum within 15 min. This approach was performed on nitrocellulose (NC) membrane assembled LFIA strips via indirect competitive immunoassay and exhibited a wide linear range of detection (0.1–80 μg*ml−1) with a low limit of detection (69.2 ng*ml−1). The coefficients of variation (CV) of the intra- and inter-assay in the detection of VAN were 7.12–8.53% and 8.46–11.82%, respectively. The dilution test and specificity indicated this method had a stability that was not affected by the serum matrix and some other antibiotics. Furthermore, the applicability of the proposed method was assessed by comparing the determined results with those measured by LC-MS/MS, showing a satisfactory correlation (R2 = 0.9713). The proposed CN-EUs-based LFIA manifested promising analytical performance, which showed potential value in the real-time monitoring of VAN and could help optimize the clinical use of more antibiotics.

Sign in / Sign up

Export Citation Format

Share Document