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Rapid detection of Chattonella marina by PCR combined with dot lateral flow strip.

ABSTRACT: In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored. For this purpose, using Chattonella marina as a test algal species, PCR targeting the D1-D2 region of large subunit ribosomal gene of this alga was performed with the tagged specific primers. The amplicons were then analyzed with the manually prepared dot lateral flow strip, and the strip could produce a test dot and a control dot that are naked-eye detectable, indicating the successful establishment of PCDS. The established PCDS assay does not require expensive instruments for the detection, and the results can be observed visually after adding 7.5 μL of PCR amplicons in combination with 92.5 μL of chromatography buffer to the sample pad of the strip for about 10 min. The PCR conditions were optimized to enhance the effectiveness of detection. The cross-reactivity test with 23 microalgae species, including Chattonella marina, showed good specificity of the PCDS. The detection limit of PCDS was 1.25 × 10^-2 ng µL^-1 for genomic DNA and 10¹ cells mL^-1 for crude cell extracts, which can meet the detection needs. In summary, the PCDS proposed in this study has low cost, clear, and intuitive detection results and good specificity and sensitivity, providing a novel detection method for C. marina.

Supplementary information

The online version contains supplementary material available at 10.1007/s10811-021-02667-x.

SUBMITTER: Zhang C

PROVIDER: S-EPMC8778489 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Rapid detection of <i>Chattonella marina</i> by PCR combined with dot lateral flow strip.

Zhang Chunyun C Chen Qixin Q Liu Fuguo F Liu Yin Y Wang Yuanyuan Y Chen Guofu G

Journal of applied phycology 20220121 1

In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored. For this purpose, using <i>Chattonella marina</i> as a test algal species, PCR targeting the D1-D2 region of large subunit ribosomal gene of this alga was performed with the tagged specific primers. The amplicons were then analyzed with the manually prepared dot lateral flow strip, and the strip could produce a test do ...[more]

PMID: 35079200

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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious and concealed virus that causes pneumonia, severe acute respiratory syndrome, and even death. Although the epidemic has been controlled since the development of vaccines and quarantine measures, many people are still infected, particularly in third-world countries. Several methods have been developed for detection of SARS-CoV-2, but owing to its price and efficiency, the immune strip could be a better method for the third-world countries.MethodsIn this study, two antibodies were linked to latex microspheres, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, as the bridge to decrease the cost further and improve the detection performance. The specificity of the lateral flow immunoassay strip (LFIA) was tested by several common viruses and respiratory bacterial infections. Besides, the reproducibility and stability of the LFIAs were tested on the same batch of test strips. Under optimal conditions, the sensitivity of LFIA was determined by testing different dilutions of the positive specimens.ResultsThe proposed LFIAs were highly specific, and the limit of detection was as low as 25 ng/mL for SARS-CoV-2 antigens. The clinical applicability was evaluated with 659 samples (230 positive and 429 negative samples) by using both LFIA and rRT-PCR. Youden's index (J) was used to assess the performance of these diagnostic tests. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. In addition, the consistency of our proposed LFIA was analyzed using Cohen's kappa coefficient (κ = 0.9620).ConclusionWe found disease stage, age, gender, and clinical manifestations have only a slight influence on the diagnosis. Therefore, the lateral flow immunoassay SARS-CoV-2 antigen test strip is suitable for point-of-care detection and provides a great application for SARS-CoV-2 epidemic control in the third-world countries.