Use of biochemical miniaturized galleries, rRNA based lateral flow assay and Real Time PCR for Cronobacter spp. confirmation

Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.

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Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Biosensors ◽

10.3390/bios12010011 ◽

2021 ◽

Vol 12 (1) ◽

pp. 11

Author(s):

Zhijian Yi ◽

Jean de Dieu Habimana ◽

Omar Mukama ◽

Zhiyuan Li ◽

Nelson Odiwuor ◽

...

Keyword(s):

Real Time ◽

Early Stage ◽

Limited Resource ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Capture Probe ◽

Fluorescence Detector ◽

Lateral Flow Strip ◽

Global Pandemic ◽

Novel Approach

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

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Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00110-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2103-2108 ◽

Cited By ~ 29

Author(s):

Gemma L. Johnson ◽

Shah-Jalal Sarker ◽

Francesco Nannini ◽

Arianna Ferrini ◽

Emma Taylor ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Real Time ◽

Antifungal Therapy ◽

Real Time Pcr ◽

Invasive Pulmonary Aspergillosis ◽

Lateral Flow ◽

Pulmonary Aspergillosis ◽

Lateral Flow Device ◽

Flow Device ◽

The Impact

Clinical experience with the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in hemato-oncology. Invasive pulmonary aspergillosis (IPA) is the most common manifestation, so we evaluated biomarkers in bronchoalveolar lavage (BAL) fluid. AnAspergillus-specific lateral-flow device (LFD), quantitative real-time PCR (qPCR), and the galactomannan (GM) test were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven IPA, 3 had probable IPA, 6 had possible IPA, and 15 patients had no IPA by European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (EORTC/MSG) criteria. The diagnostic accuracies of the tests were evaluated, and pairwise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified to be the most useful for IPA diagnosis. Using the EORTC/MSG criteria, the sensitivities of qPCR and LFD were 100% and the sensitivity of the GM test was 87.5% (GM test index cutoff, >0.8), with the tests having specificities of between 66.7 and 86.7%. The agreement between the results of qPCR and LFD was almost perfect (Cohen's kappa coefficient = 0.93, 95% confidence interval, 0.81 to 1.00). LFD and qPCR combined had a sensitivity of 100% and a specificity of 85.7%. Calcofluor staining and culture of all BAL fluid samples were negative for fungal infection. The median time from the start of mold-active antifungal therapy to the time of collection of BAL fluid was 6 days. Reversing roles and using dual testing by LFD and qPCR to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. All three tests are useful for the diagnosis of IPA in BAL fluid samples. Despite the significant delays between the start of antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remained informative. In particular, the combination of LFD and qPCR allows the sensitive and specific detection of IPA.

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Evaluation of Real-Time PCR, Galactomannan Enzyme-Linked Immunosorbent Assay (ELISA), and a Novel Lateral-Flow Device for Diagnosis of Invasive Aspergillosis

Journal of Clinical Microbiology ◽

10.1128/jcm.03189-12 ◽

2013 ◽

Vol 51 (5) ◽

pp. 1510-1516 ◽

Cited By ~ 106

Author(s):

P. L. White ◽

C. Parr ◽

C. Thornton ◽

R. A. Barnes

Keyword(s):

Invasive Aspergillosis ◽

Real Time ◽

Immunosorbent Assay ◽

Real Time Pcr ◽

Lateral Flow ◽

Enzyme Linked Immunosorbent Assay ◽

Lateral Flow Device ◽

Flow Device

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

10.21203/rs.3.rs-17865/v2 ◽

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.

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Development of loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay for Mycoplasma pneumoniae detection

AMB Express ◽

10.1186/s13568-019-0921-3 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Yacui Wang ◽

Yi Wang ◽

Weiwei Jiao ◽

Jieqiong Li ◽

Shuting Quan ◽

...

Keyword(s):

Tract Infection ◽

Respiratory Tract ◽

Respiratory Tract Infection ◽

Real Time ◽

Mycoplasma Pneumoniae ◽

Real Time Pcr ◽

Lateral Flow ◽

Isothermal Amplification ◽

Dna Templates ◽

Loop Mediated Isothermal Amplification

AbstractMycoplasma pneumoniae (MP) is one of the most common pathogens causing respiratory tract infection, especially for community-acquired pneumonia (CAP) in school-age children. There was considerable amount of studies on loop-mediated isothermal amplification (LAMP) assay for MP detection. However, the result interpretation of these developed LAMP assays was sophisticated and subjective. Therefore, we developed and evaluated a LAMP coupled with nanoparticle-based lateral flow biosensor (LFB) assay (LAMP-LFB) for simple, reliable, and objective identification of MP (MP-LAMP-LFB). Six primers specific to P1 gene of MP were designed, and the preferred temperature for this assay was confirmed to be 65 °C. The amplification products could be visually interpreted by LFB within 2 min. The MP-LAMP-LFB assay specifically identified DNA templates of MP, and no cross-reactivity with other pathogens was obtained. The limit of the detection for this assay was 600 fg of DNA templates in pure cultures, which was in complete accordance with colorimetric indicator detection and agarose gel electrophoresis analysis. This assay was applied to 209 oropharyngeal swab specimens collected from children with acute respiratory tract infection for clinical evaluation, and compared to real-time PCR detection. Using the LAMP-LFB and real-time PCR assay, the positive rates of MP were 47.8% and 31.6%, respectively. Results suggested that the LAMP-LFB assay displayed high sensitivity compared to real-time PCR method. In summary, LAMP-LFB assay established here was a simple, objective, and sensitive assay for MP detection, which can be widely applied in clinical settings, especially in rural areas.

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Faculty Opinions recommendation of Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725447052.793528004 ◽

2017 ◽

Author(s):

Stéphane Bretagne

Keyword(s):

Bronchoalveolar Lavage ◽

Real Time ◽

Clinical Diagnosis ◽

Real Time Pcr ◽

Bronchoalveolar Lavage Fluid ◽

Invasive Pulmonary Aspergillosis ◽

Lavage Fluid ◽

Lateral Flow ◽

Pulmonary Aspergillosis ◽

Flow Device

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

10.21203/rs.3.rs-17865/v1 ◽

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. It is an urgent need to develop a rapid and accurate method to detect M. ovipneumoniae . 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 16SrRNA 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 pathogens tested, especially the M. capricolum subsp. capripneumoniae . 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 higher than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 46 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 17 samples in the RPA assays and 19 samples in the real-time PCR assay. The real-time RPA and LFS RPA showed diagnostic specificity of 100%, diagnostic sensitivity of 89.47%, and a kappa coefficient of 0.909. 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.

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