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 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 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 Rapid detection of Clostridium perfringens in food by loop-mediated isothermal amplification combined with a lateral flow biosensor

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245144
Author(s):  
Thanawat Sridapan ◽  
Wanida Tangkawsakul ◽  
Tavan Janvilisri ◽  
Wansika Kiatpathomchai ◽  
Sirintip Dangtip ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Culture Method ◽  
Food Samples ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Bacterial Strains ◽  
Commercial Test ◽  
Loop Mediated Isothermal Amplification ◽  
Test Kit

Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection by standard culture method is time-consuming and labor intensive. Current rapid commercial test kits are prohibitively expensive. It is thus necessary to develop rapid and cost-effective detection tool. Here, loop-mediated isothermal amplification (LAMP) in combination with a lateral-flow biosensor (LFB) was developed for visual inspection of C. perfringens-specific cpa gene. The specificity of the developed test was evaluated against 40 C. perfringens and 35 other bacterial strains, which showed no cross-reactivity, indicating 100% inclusivity and exclusivity. LAMP-LFB detection limit for artificially contaminated samples after enrichment for 16 h was 1–10 CFU/g sample, which was comparable to the commercial real-time PCR kit. The detection performance of LAMP-LFB was also compared to culture-based method using 95 food samples, which revealed the sensitivity (SE), specificity (SP) and Cohen's kappa coefficient (κ) of 88.0% (95% CI, 75.6%-95.4%), 95.5% (95% CI, 84.8%-99.4%) and 0.832 (95% CI, 0.721–0.943), respectively. Area under the receiver operating characteristic (ROC) curve was 0.918 (95% CI, 0.854–0.981), indicating LAMP-LFB as high relative accuracy test. In conclusion, LAMP-LFB assay is a low-cost qualitative method and easily available for routine detection of C. perfringens in food samples, which could serve as an alternative to commercial test kit.

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 Development of a Nucleic Acid Lateral Flow Immunoassay for the Detection of Human Polyomavirus BK

Diagnostics ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 403 ◽  
Author(s):  
Yi-Huei Huang ◽  
Kuan-Yi Yu ◽  
Shou-Ping Huang ◽  
Hui-Wen Chuang ◽  
Wen-Zhi Lin ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Jc Virus ◽  
Point Of Care ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cross Reactivity ◽  
Bk Virus ◽  
Lateral Flow ◽  
Human Polyomavirus ◽  
Lateral Flow Immunoassay

The BK virus (BKV) is an emerging pathogen in immunocompromised individuals and widespread in the human population. Polymerase chain reaction is a simple and highly sensitive method for detecting BKV, but it is time consuming and requires expensive instruments and expert judgment. The lateral flow assay, a rapid, low-cost, minimal-labor, and easy-to-use diagnostic method, was successfully applied for pathogen detection. In this study, we used oligonucleotide probes to develop a simple and rapid sandwich-type lateral flow immunoassay for detecting BKV DNA within 45 minutes. The detection limit for the synthetic single-stranded DNA was 5 nM. The specificity study showed no cross-reactivity with other polyomaviruses, such as JC virus and simian virus 40. For the Escherichia coli containing BKV plasmid cultured samples, the sensitivity was determined to be 107 copies/mL. The approach offers great potential for BKV detection of various target analytes in point-of-care settings.

scholarly journals Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 762
Author(s):  
Yihong He ◽  
Wenxian Chen ◽  
Jindai Fan ◽  
Shuangqi Fan ◽  
Hongxing Ding ◽  
...  
Keyword(s):  
Detection Method ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Porcine Parvovirus ◽  
Swine Industry ◽  
Resource Limited ◽  
Efficient Detection ◽  
Lateral Flow Dipstick ◽  
Polymerase Chain ◽  
Embryonic Death

Porcine parvovirus (PPV) infection is the primary cause of SMEDI (stillbirth; mummification; embryonic death; infertility) syndrome, which is a global burden for the swine industry. Thus, it is crucial to establish a rapid and efficient detection method against PPV infection. In the present work, we developed a recombinase-aided amplification (RAA) assay, coupled with a lateral flow dipstick (LFD), to achieve an amplification of PPV DNA at 37 °C within 15 min. The detection limits of PPV RAA-LFD assay were 102 copies/μL recombinant plasmid pMD19-T-VP1, 6.38 × 10−7 ng/μL PPV DNA, and 10−1 TCID50/mL virus, respectively. This method was highly specific for PPV detection with no cross-reactivity for other swine pathogens. In contrast to polymerase chain reaction (PCR), the PPV RAA-LFD assay is more sensitive and cost-saving. Hence, the established PPV RAA-LFD assay provided an alternative for PPV detection, especially in resource-limited regions.

scholarly journals Visual multiple cross displacement amplification for the rapid identification of S. agalactiae immediately from vaginal and rectal swabs

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xueqin Cheng ◽  
Zhiqian Dou ◽  
Jing Yang ◽  
Dexi Liu ◽  
Yulong Gu ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Clinical Specimen ◽  
Rapid Identification ◽  
Sample Collection ◽  
Special Equipment ◽  
Multiple Cross ◽  
Rectal Swabs ◽  
Cost Efficient ◽  
The Cost ◽  
Positive Results

AbstractStreptococcus agalactiae (S. agalactiae) is an important pathogen that can lead to neonatus and mother infection. The current existing techniques for the identification of S. agalactiae are limited by accuracy, speed and high-cost. Therefore, a new multiple cross displacement amplification (MCDA) assay was developed for test of the target pathogen immediately from vaginal and rectal swabs. MCDA primers screening were conducted targeting S. agalactiae pcsB gene, and one set of MCDA primers with better rapidity and efficiency was selected for establishing the S. agalactiae-MCDA assay. As a result, the MCDA method could be completed at a constant temperature of 61 °C, without the requirement of special equipment. The detection limit is 250 fg (31.5 copies) per reaction, all S. agalactiae strains displayed positive results, but not for non-S. agalactiae strains. The visual MCDA assay detected 16 positive samples from 200 clinical specimen, which were also detected positive by enrichment/qPCR. While the CHROMagar culture detected 6 positive samples. Thus, the MCDA assay is prefer to enrichment/qPCR and culture for detecting S. agalactiae from clinical specimen. Particularly, the whole test of MCDA takes about 63.1 min, including sample collection (3 min), DNA preparation (15 min), MCDA reaction (45 min) and result reporting (6 s). In addition, the cost was very economic, with only US$ 4.9. These results indicated that our S. agalaciae-MCDA assay is a rapid, sensitive and cost-efficient technique for target pathogen detection, and is more suitable than conventional assays for an urgent detection, especially for 'on-site' laboratories and resource-constrained settings.

scholarly journals Rapid Antibody Selection Using Surface Plasmon Resonance for High-Speed and Sensitive Hazelnut Lateral Flow Prototypes

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 130 ◽  
Author(s):  
Georgina Ross ◽  
Maria Bremer ◽  
Jan Wichers ◽  
Aart van Amerongen ◽  
Michel Nielen
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
High Speed ◽  
Selection Process ◽  
Low Cost ◽  
Real Life ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Label Free

Lateral Flow Immunoassays (LFIAs) allow for rapid, low-cost, screening of many biomolecules such as food allergens. Despite being classified as rapid tests, many LFIAs take 10–20 min to complete. For a really high-speed LFIA, it is necessary to assess antibody association kinetics. By using a label-free optical technique such as Surface Plasmon Resonance (SPR), it is possible to screen crude monoclonal antibody (mAb) preparations for their association rates against a target. Herein, we describe an SPR-based method for screening and selecting crude anti-hazelnut antibodies based on their relative association rates, cross reactivity and sandwich pairing capabilities, for subsequent application in a rapid ligand binding assay. Thanks to the SPR selection process, only the fast mAb (F-50-6B12) and the slow (S-50-5H9) mAb needed purification for labelling with carbon nanoparticles to exploit high-speed LFIA prototypes. The kinetics observed in SPR were reflected in LFIA, with the test line appearing within 30 s, almost two times faster when F-50-6B12 was used, compared with S-50-5H9. Additionally, the LFIAs have demonstrated their future applicability to real life samples by detecting hazelnut in the sub-ppm range in a cookie matrix. Finally, these LFIAs not only provide a qualitative result when read visually, but also generate semi-quantitative data when exploiting freely downloadable smartphone apps.

scholarly journals Potential Antigenic Cross-reactivity Between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Dengue Viruses

2020 ◽  
Author(s):  
Yaniv Lustig ◽  
Shlomit Keler ◽  
Rachel Kolodny ◽  
Nir Ben-Tal ◽  
Danit Atias-Varon ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Envelope Protein ◽  
False Positive ◽  
In Silico ◽  
Rapid Test ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Testing

Abstract Background Coronavirus disease 2019 (COVID-19) and dengue fever are difficult to distinguish given shared clinical and laboratory features. Failing to consider COVID-19 due to false-positive dengue serology can have serious implications. We aimed to assess this possible cross-reactivity. Methods We analyzed clinical data and serum samples from 55 individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To assess dengue serology status, we used dengue-specific antibodies by means of lateral-flow rapid test, as well as enzyme-linked immunosorbent assay (ELISA). Additionally, we tested SARS-CoV-2 serology status in patients with dengue and performed in-silico protein structural analysis to identify epitope similarities. Results Using the dengue lateral-flow rapid test we detected 12 positive cases out of the 55 (21.8%) COVID-19 patients versus zero positive cases in a control group of 70 healthy individuals (P = 2.5E−5). This includes 9 cases of positive immunoglobulin M (IgM), 2 cases of positive immunoglobulin G (IgG), and 1 case of positive IgM as well as IgG antibodies. ELISA testing for dengue was positive in 2 additional subjects using envelope protein directed antibodies. Out of 95 samples obtained from patients diagnosed with dengue before September 2019, SARS-CoV-2 serology targeting the S protein was positive/equivocal in 21 (22%) (16 IgA, 5 IgG) versus 4 positives/equivocal in 102 controls (4%) (P = 1.6E−4). Subsequent in-silico analysis revealed possible similarities between SARS-CoV-2 epitopes in the HR2 domain of the spike protein and the dengue envelope protein. Conclusions Our findings support possible cross-reactivity between dengue virus and SARS-CoV-2, which can lead to false-positive dengue serology among COVID-19 patients and vice versa. This can have serious consequences for both patient care and public health.

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