scholarly journals Establishment and Application of Multiple Cross Displacement Amplification Coupled With Lateral Flow Biosensor (MCDA-LFB) for Visual and Rapid Detection of Candida albicans in Clinical Samples

2019 ◽  
Vol 9 ◽  
Author(s):  
Fan Zhao ◽  
Lina Niu ◽  
Linlin Yan ◽  
Jinqing Nong ◽  
Chunmei Wang ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Rapid Detection ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Multiple Cross

scholarly journals Rapid Detection of Aspergillus fumigatus Using Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Lateral Flow

2021 ◽  
Vol 11 ◽  
Author(s):  
Luxi Jiang ◽  
Xiaomeng Li ◽  
Rumeng Gu ◽  
Deguang Mu
Keyword(s):  
Aspergillus Fumigatus ◽  
Detection Method ◽  
Culture Method ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Specific Detection ◽  
Minimum Concentration ◽  
Whole Process ◽  
Multiple Cross ◽  
Pcr Method

Aspergillus fumigatus is an opportunistic, ubiquitous, saprophytic mold which can cause infection in the lungs, nose, eyes, brain, and bones in humans, especially in immunocompromised patients. However, it is difficult to diagnose A. fumigatus infection quickly. Here, we introduce a new detection method, namely multiple cross displacement amplification (MCDA) combined with nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB), which was proved to be fast, reliable, and simple for detecting A. fumigatus. We designed a set of 10 primers targeting the gene annexin ANXC4 of A. fumigatus. The best MCDA condition is 66 °C for 35 min. The minimum concentration that can be detected by this method was 10 fg. In the case of 100 sputum samples, 20 (20%) and 15 (15%) samples were positive by MCDA-LFB and PCR method, respectively. MCDA-LFB and traditional culture method showed the same results. Compared with the culture method, the diagnostic accuracy of MCDA-LFB can reach 100%. It showed that the MCDA-LFB method has better detection ability than the PCR method. We found that the whole process could be controlled within 60 min including the preparation of DNA (20 min), MCDA reaction (35 min) and results reporting (2 min). These results show that this assay is suitable for the rapid, sensitive and specific detection of A. fumigatus in clinical samples.

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 Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

2020 ◽  
Author(s):  
Thomas R Shelite ◽  
Ashanti C Uscanga-Palomeque ◽  
Alejandro Castellanos ◽  
Peter C Melby ◽  
Bruno L Travi
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
The United States ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Reference Test ◽  
Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

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 Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

2020 ◽  
Author(s):  
Thomas R Shelite ◽  
Ashanti C Uscanga-Palomeque ◽  
Alejandro Castellanos ◽  
Peter C Melby ◽  
Bruno L Travi
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
The United States ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Reference Test ◽  
Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

scholarly journals Development and Application of a Multiple Cross Displacement Amplification Combined With Nanoparticle-Based Lateral Flow Biosensor Assay to Detect Candida tropicalis

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu Wang ◽  
Xue Zhao ◽  
Jinzhi Cheng ◽  
Xiaomin Tang ◽  
Xu Chen ◽  
...  
Keyword(s):  
Candida Tropicalis ◽  
Gold Standard ◽  
Color Change ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Reaction Products ◽  
Special Equipment ◽  
Gold Standard Method ◽  
Study Results ◽  
Multiple Cross

Candida tropicalis is an increasingly opportunistic pathogen that causes serious invasive candidiasis threatening a patient’s life. Traditional methods to detect C. tropicalis infection depends on time-consuming, culture-based gold-standard methods. So, we sought to establish a new method that could detect target pathogens quickly, accurately, and straightforwardly. Herein, a combination of multiple cross displacement amplification (MCDA) and lateral flow biosensors (LFB) was employed to detect C. tropicalis. In the MCDA system, 10 primers were designed to identify the specific genes of C. tropicalis and amplify the genes in an isothermal amplification device. Then, MCDA amplification reaction products could be identified visibly by color change, and all the amplification products would be tested by LFB with no special equipment. The results demonstrated that the optimal reaction condition of C. tropicalis-MCDA assay was 64°C within 30 min, and only 10 fg DNA was required in each reaction. No cross-reaction was found between C. tropicalis strains and non-C. tropicalis strains. For 300 sputum samples, the results showed that MCDA-LFB assay could rapidly and successfully detect all of the C. tropicalis-positive (28/300) samples detected by the gold-standard method. The entire procedure, including specimen processing (40 min), isothermal reaction (30 min) and result reporting (within 2 min), could be completed within 75 min. Briefly, the study results demonstrated that the detection ability of C. tropicalis-MCDA-LFB assay was better than culture methods with more simplicity, rapidity, sensitivity and specificity. Hence, MCDA-LFB strategy is an effective tool to rapidly detect C. tropicalis in clinical samples, especially in resource-poor areas.

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 immunochromatographic lateral flow test for rapid detection of Clostridium perfringens α, β and ε toxins in clinical samples

2021 ◽  
Vol 24 (4) ◽  
pp. 497-507
Author(s):  
R Soliman ◽  
M. M. Magdy ◽  
A. Samir ◽  
Y. A. Abdalla ◽  
R. H. Sayed
Keyword(s):  
Guinea Pigs ◽  
Rapid Detection ◽  
Polyclonal Antibodies ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Immunochromatographic Test ◽  
Flow Test ◽  
Faecal Samples ◽  
Lateral Flow Test ◽  
Sensitivity Specificity

In the present work a lateral flow immunochromatographic test (LFT) for rapid detection of Clostri­dium perfringens toxins types, alpha (α), beta (β) and epsilon (ε) in clinical samples was developed. C. perfringens toxins were prepared, purified and inactivated with 0.2% formalin. Polyclonal antibodies specific to C. perfringens toxins types α, β and ε toxoids were prepared in rabbits and guinea pigs. The toxoid specific polyclonal antibodies prepared in rabbits were labelled with gold chloride nanoparticles. The prepared toxin specific rabbit and guinea pigs antibodies and goat anti-rabbit antibodies were utilised in development of a lateral flow immunochromatographic test and the latter - evaluated for detection of C. perfringens α, β and ε toxins in clinical samples. The sensitivity and specificity and accuracy of the developed LFT were determined by comparison with a commercially available ELISA used for detection of these toxins. The prepared LFT was capable to detect C. perfringens α, β and ε toxins in quantities of 2 μg/ml, 250 ng/ml and 60 ng/ml, respectively. One hundred poultry suspected faecal samples was examined both with the prepared LFT and commercial ELISA to test the validity of developed LFT. The sensitivity, specificity and accuracy of the LFT for detection of C. perfringens toxins were 81%, 95.2% and 90%, respectively, for α toxin, 76.6%, 98.5% and 72%, respectively, for β toxin and 66.6%, 98.8% and 95%, respectively, for ε toxin.

Sign in / Sign up

Export Citation Format

Share Document