Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
Background: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplific...
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| Format: | Article |
| Language: | English |
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Eco-vector
2021-10-01
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| Series: | Клиническая практика |
| Subjects: | |
| Online Access: | https://journals.eco-vector.com/clinpractice/article/viewFile/78139/pdf |
| _version_ | 1797721918126686208 |
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| author | Anna K. Shuryaeva Tatyana V. Malova Anna A. Tolokonceva Sofia A. Karseka Ekaterina E. Davydova German A. Shipulin |
| author_facet | Anna K. Shuryaeva Tatyana V. Malova Anna A. Tolokonceva Sofia A. Karseka Ekaterina E. Davydova German A. Shipulin |
| author_sort | Anna K. Shuryaeva |
| collection | DOAJ |
| description | Background: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplification assay will allow simplifying the procedure and reducing the time of detection for a bedside application.
Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with a fluorescent probe for the diagnosis of campylobacteriosis.
Methods: Stool suspensions were prepared and bacterial fractions were separated as described in the methodological recommendations of the Central Research Institute of Epidemiology. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturer's instruction and detected with AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation). Primers and probes were selected in a 16S rDNA gene region. Analytical specificity was confirmed on bacterial cultures, analytical sensitivity was assessed using a recombinant plasmid containing the target Campylobacter DNA sequence fragment. LAMP amplification was performed at 65C for 30 min.
Results: An assay for the detection of Campylobacter spp. based on loop-mediated isothermal amplification has been developed, the reaction time does not exceed 30 minutes. The analytical sensitivity of the developed technique is comparable to the real-time PCR and is equal to 103 copies/ml, the analytical specificity is 100%. The evaluation of 127 clinical samples, previously characterized by a commercial kit, AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation), showed high diagnostic specificity and sensitivity of the developed LAMP-method. No false positive results were found, 108 samples were negative by LAMP and PCR. Campylobacter spp. DNA was detected by the LAMP method in 18 out of 19 PCR-positive samples. One discordant LAMP negative sample can be attributed to the low bacterial load of Campylobacter spp. for a given sample. Conclusions: A method for the rapid detection of Campylobacter spp. loop-mediated isothermal amplification has been developed, and its high analytical and diagnostic characteristics have been shown experimentally. |
| first_indexed | 2024-03-12T09:40:01Z |
| format | Article |
| id | doaj.art-51e0539f37bd4259bbbcd0059720f3c3 |
| institution | Directory Open Access Journal |
| issn | 2220-3095 2618-8627 |
| language | English |
| last_indexed | 2024-03-12T09:40:01Z |
| publishDate | 2021-10-01 |
| publisher | Eco-vector |
| record_format | Article |
| series | Клиническая практика |
| spelling | doaj.art-51e0539f37bd4259bbbcd0059720f3c32023-09-02T13:18:24ZengEco-vectorКлиническая практика2220-30952618-86272021-10-01123303510.17816/clinpract7813972875Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosisAnna K. Shuryaeva0https://orcid.org/0000-0003-2227-1840Tatyana V. Malova1https://orcid.org/0000-0001-8546-1444Anna A. Tolokonceva2https://orcid.org/0000-0002-8757-081XSofia A. Karseka3https://orcid.org/0000-0002-9988-6918Ekaterina E. Davydova4https://orcid.org/0000-0003-2926-0490German A. Shipulin5https://orcid.org/0000-0002-3668-6601Centre for Strategic Planning and Management of Biomedical Health RisksCentre for Strategic Planning and Management of Biomedical Health RisksCentre for Strategic Planning and Management of Biomedical Health RisksCentre for Strategic Planning and Management of Biomedical Health RisksCentre for Strategic Planning and Management of Biomedical Health RisksCentre for Strategic Planning and Management of Biomedical Health RisksBackground: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplification assay will allow simplifying the procedure and reducing the time of detection for a bedside application. Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with a fluorescent probe for the diagnosis of campylobacteriosis. Methods: Stool suspensions were prepared and bacterial fractions were separated as described in the methodological recommendations of the Central Research Institute of Epidemiology. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturer's instruction and detected with AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation). Primers and probes were selected in a 16S rDNA gene region. Analytical specificity was confirmed on bacterial cultures, analytical sensitivity was assessed using a recombinant plasmid containing the target Campylobacter DNA sequence fragment. LAMP amplification was performed at 65C for 30 min. Results: An assay for the detection of Campylobacter spp. based on loop-mediated isothermal amplification has been developed, the reaction time does not exceed 30 minutes. The analytical sensitivity of the developed technique is comparable to the real-time PCR and is equal to 103 copies/ml, the analytical specificity is 100%. The evaluation of 127 clinical samples, previously characterized by a commercial kit, AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation), showed high diagnostic specificity and sensitivity of the developed LAMP-method. No false positive results were found, 108 samples were negative by LAMP and PCR. Campylobacter spp. DNA was detected by the LAMP method in 18 out of 19 PCR-positive samples. One discordant LAMP negative sample can be attributed to the low bacterial load of Campylobacter spp. for a given sample. Conclusions: A method for the rapid detection of Campylobacter spp. loop-mediated isothermal amplification has been developed, and its high analytical and diagnostic characteristics have been shown experimentally.https://journals.eco-vector.com/clinpractice/article/viewFile/78139/pdfgastrointestinal infectionsmolecular diagnosticsrapid diagnosticsloop-mediated isothermal amplificationlampcampylobacter spp. |
| spellingShingle | Anna K. Shuryaeva Tatyana V. Malova Anna A. Tolokonceva Sofia A. Karseka Ekaterina E. Davydova German A. Shipulin Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis Клиническая практика gastrointestinal infections molecular diagnostics rapid diagnostics loop-mediated isothermal amplification lamp campylobacter spp. |
| title | Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis |
| title_full | Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis |
| title_fullStr | Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis |
| title_full_unstemmed | Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis |
| title_short | Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis |
| title_sort | development and evaluation of a loop mediated isothermal amplification assay lamp for the diagnosis of campylobacteriosis |
| topic | gastrointestinal infections molecular diagnostics rapid diagnostics loop-mediated isothermal amplification lamp campylobacter spp. |
| url | https://journals.eco-vector.com/clinpractice/article/viewFile/78139/pdf |
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