Diagnostic method for biological sample, diagnostic kit for biological sample, and oligonucleotide

By using the LAMP assay and species-specific primer sets in the diagnosis of leishmaniasis, combined with colorimetric or fluorescence methods and gold nanorod detection, the problems of insufficient sensitivity and specificity in existing technologies are solved, and rapid and accurate identification of Leishmania species is achieved.

CN120659887APending Publication Date: 2025-09-16OSVALDO CRUZ FOUNDATION
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Patent Information

Application Number
CN202380093160.2
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Priority Date
2022-12-27
Filing Date
2023-12-22
Publication Date
2025-09-16

AI Technical Summary

Technical Problem

Existing diagnostic technologies for leishmaniasis lack sensitivity and specificity, especially parasitological and immunological detection methods are prone to cross-reactions, and molecular detection requires complex laboratory equipment, making it difficult to be widely used in primary health units.

Method used

The loop-mediated isothermal amplification (LAMP) assay in single or multiplex format is used to detect Leishmania species using species-specific primer sets, combined with colorimetric or fluorescent visualization, and gold nanorods as biosensors for amplification product detection.

Benefits of technology

It has achieved rapid and accurate identification of Leishmania species in primary health units, reduced detection time and dependence on laboratory equipment, and improved detection sensitivity and specificity.

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Abstract

The invention provides a diagnosis method, in particular to a leishmaniasis diagnosis method based on a multiple LAMP (loop-mediated isothermal amplification) detection method. In addition, the present invention provides a diagnostic kit for a biological sample and an oligonucleotide for use in the method.
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Claims

1. A method for diagnosing a biological sample, wherein: The method comprises: (i) performing loop-mediated isothermal amplification (LAMP) on the sample using a primer set specific for the target nucleic acid, wherein the primer set comprises at least four primers selected from the group consisting of F3, B3, FIP, BIP, LF and LB; (ii) Detecting the target nucleic acid amplification product in the biological sample.

2. The method according to claim 1, wherein The biological sample is from a mammalian individual.

3. The method according to claim 1 or 2, wherein: In step (i), the target nucleic acid is a nucleic acid from a flagellated protozoan belonging to the Trypanosomatidae family and the Leishmania genus.

4. The method according to any one of claims 1 to 3, wherein The LAMP reaction is carried out at a temperature in the range of about 60 to 70°C.

5. The method according to any one of claims 1 to 4, wherein The primer set comprises primers whose sequences are at least 90% identical to the following sequences: the sequences shown in SEQ ID NOs: 1 to 8 for the amazonensis species; the sequences shown in SEQ ID NOs: 9 to 16 for the brazilian species; and the sequences shown in SEQ ID NOs: 17 to 23 for the infantum species.

6. The method according to claim 5, wherein: The primer set for colorimetric development comprises primers whose sequences are at least 90% identical to the following sequences: the sequences shown in SEQ ID NOs: 1 to 6 for the amazonensis species; the sequences shown in SEQ ID NOs: 9 to 14 for the brazilian species; and the sequences shown in SEQ ID NOs: 17 to 22 for the infantum species.

7. The method according to claim 5, wherein: The primer set for fluorescent color development comprises primers whose sequences are at least 90% identical to the following sequences: the sequences shown in SEQ ID NO: 1 to 5, 7 or 8 for the amazonensis species; the sequences shown in SEQ ID NO: 9 to 13, 15 or 16 for the brazilian Leishmania species; and the sequences shown in SEQ ID NO: 17 to 20, 22 and 23 for the infant Leishmania species.

8. The method according to claim 7, wherein: The method further comprises adding a quencher having a sequence at least 90% identical to the sequence shown in SEQ ID NO:

24.

9. The method according to claim 7, wherein: The detection of the color change of the amplification product is performed with the aid of a biosensing material consisting of gold nanorods functionalized with a probe sequence comprising SEQ ID NO: 25 for Leishmania amazonensis or SEQ ID NO: 26 for Leishmania braziliensis.

10. A diagnostic kit for a biological sample, comprising a primer set, wherein the primer set comprises: 6 primers, each primer having a sequence that is at least 90% identical to SEQ ID NOs: 1 to 23, respectively.

11. An oligonucleotide comprising a sequence shown in SEQ ID NOs: 1 to 23, 25 and 26 or a sequence having 90% identity thereto.