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 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 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 A Rapid Detection of Haemophilus influenzae Using Multiple Cross Displacement Amplification Linked With Nanoparticle-Based Lateral Flow Biosensor

2021 ◽  
Vol 11 ◽  
Author(s):  
Qilong Cao ◽  
Shaoshuai Liang ◽  
Lin Wang ◽  
Jun Cao ◽  
Mengyang Liu ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Pathogen Detection ◽  
Traditional Culture ◽  
Culture Method ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Pcr Detection ◽  
Minimum Concentration ◽  
Human Pathogenic Bacterium ◽  
Multiple Cross

Haemophilus influenzae is a major human pathogenic bacterium, resulting in a series of diseases, such as pneumonia, bacteremia, meningitis. However, it is hard to diagnose H. influenzae quickly. In this study, the multiple cross displacement amplification (MCDA) and nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB) were combined to detect H. influenzae, which has been proven to be reliable, rapid, and not complicated. On the basis of H. influenzae outer membrane protein P6 gene, 10 specific primers were designed. The best MCDA condition was 61°C for 1 h. The sensitivity of H. influenzae-MCD-LFB assay showed, in the pure cultures, the minimum concentration of genomic DNA templates was 100 fg. The specificity of H. influenzae-MCD-LFB assay showed only H. influenzae templates were detected, and no cross-reactivity was found in non-H. influenzae isolates and other Haemophilus species. In 56 sputum samples, with MCDA-LFB method and PCR detection, 21 samples were positive, which was in consistent with the traditional culture method. The accuracy of diagnosis of MCDA-LFB, in comparison with the traditional culture method and PCR detection, can reach 100%, indicating that the MCDA-LFB assay gains an advantage over the cultured-based method for target pathogen detection. In conclusion, the MCDA-LFB assay is suitable for the sensitive, rapid, and specific detection of H. influenzae, which might be used as a potential diagnostic tool for H. influenzae in basic and clinical laboratories.

scholarly journals Comparison of Updated Methods for Legionella Detection in Environmental Water Samples

2021 ◽  
Vol 18 (10) ◽  
pp. 5436
Author(s):  
Daniela Toplitsch ◽  
Sabine Platzer ◽  
Romana Zehner ◽  
Stephanie Maitz ◽  
Franz Mascher ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Limit Of Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Microbial Flora ◽  
Culture Methods ◽  
Outbreak Investigations ◽  
Standard Culture ◽  
Selection Of

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites.

scholarly journals Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci

2020 ◽  
Vol 76 (1) ◽  
pp. 146-151 ◽  
Author(s):  
Saoussen Oueslati ◽  
Hervé Volland ◽  
Vincent Cattoir ◽  
Sandrine Bernabeu ◽  
Delphine Girlich ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Culture Medium ◽  
Limit Of Detection ◽  
Culture Media ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Clinical Microbiology Laboratory ◽  
Mueller Hinton ◽  
Reliable Detection ◽  
Development And Validation

Abstract Background VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Objectives To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. Methods NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller–Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. Results All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. Conclusions NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

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 Lateral flow biosensor combined with loop-mediated isothermal amplification for detection of legionella pneumophila.

2021 ◽  
Author(s):  
Luxi Jiang ◽  
Xiaomeng Li ◽  
Rumeng Gu ◽  
Ziling Shi ◽  
Meijun Song ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Culture Method ◽  
Lavage Fluid ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Specific Gene ◽  
Minimum Concentration ◽  
Loop Mediated Isothermal Amplification ◽  
Whole Process

Abstract Legionella pneumophila ( L. pneumophila ) is the most pathogenic species of Legionella , which can cause Legionella disease. It can cause pneumonia, or Pontiac fever. In severe cases, it can lead to respiratory failure and kidney failure, with a high fatality rate. Here, a novel molecular diagnosis method, a loop-mediated isothermal amplification coupled with lateral flow biosensor (LFB) method (LAMP-LFB) was successfully established and evaluated for the identification of L. pneumophila . A set of 6 primers was designed specifically based on the L. pneumophila -specific gene mip. The optimized time and temperature conditions for the LAMP was 50 min and 64◦C respectively. The minimum concentration that can be detected by this method was 100fg. Using the protocol, we could observe the LAMP amplification within 2min by LFB. The whole process, including the preparation of DNA (20 min), LAMP reaction (50 min) and results reporting (2 min), could be finished within 75 min. Among 50 alveolar lavage fluid samples, 5(10%) were L. pneumophila -positive by the LAMP-LFB, and the diagnostic accuracy was 100% when compared to the culture method. While only 4 samples were positive using PCR method. In a word, the LAMP-LFB assay is a rapid, sensitive and specific detection method that can detect Legionella pneumophila , and it can be used as a new molecular method for the detection of target pathogens in water, environmental and clinical samples.

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 Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

2005 ◽  
Vol 71 (7) ◽  
pp. 4086-4096 ◽  
Author(s):  
Pilar Delgado-Viscogliosi ◽  
Tristan Simonart ◽  
Virginie Parent ◽  
Grégory Marchand ◽  
Marie Dobbelaere ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Limit Of Detection ◽  
Culture Method ◽  
Epifluorescence Microscopy ◽  
Specific Method ◽  
Double Staining ◽  
Culture Methods ◽  
Experimental Conditions ◽  
Short Period ◽  
Standard Culture

ABSTRACT A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.

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