# US 12,110,563 B1 · Multiple lamp primer set, detection method, and kit for simultaneous detection of multiple pathogens

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- Number: US 12,110,563 B1
- Document ID: US12110563B1
- Status: Granted
- Kind: B1
- Application number: 18/425,327
- Filed: 2024-01-29
- Granted: 2024-10-08
- Earliest claimed: 2023-05-29
- Assignees: Hainan University
- Applicants: Hainan University
- Inventors: Yun Sun; Zhenjie Cao; Yongcan Zhou; Tao Li; Ying Wu; Hehe Du; Weiliang Guo; Shifeng Wang
- Web page: https://patentgrep.com/patent/US12110563B1

## CPC

- C12Q 1/689: for bacteria
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/701: {Specific hybridization probes}
- C12Q 2600/16: Primer sets for multiplex assays
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present disclosure relates to a multiple LAMP primer set, a detection method, and a kit for simultaneously detecting multiple pathogens, belonging to the field of microbial detection. The multiple pathogens are Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus. The detection primer set is as shown in SEQ ID NO.: 1-18. The present disclosure further provides an application, a kit, and a detection method of multiple LAMP primer sets in simultaneously detecting multiple pathogens. The method of the present disclosure can simultaneously detect whether a sample contains the Vibrio harveyi, the Vibrio parahaemolyticus, or the Singapore grouper iridovirus, has strong specificity, convenient and simple operation, and has no need for an expensive instrument and equipment.

## Claims

### Claim 1

A multiple LAMP primer set that simultaneously detects multiple pathogens, wherein the multiple pathogens are Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus; the multiple LAMP primer set is shown in SEQ ID NO.: 1-18.

### Claim 2 (depends on 1)

A detection method for the multiple LAMP primer set as claimed in claim 1, wherein the detection method simultaneously detects multiple pathogens and is for non-disease diagnosis and non-treatment purposes, the multiple pathogens are Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus; the detection method comprises the following steps:
(1) collecting a sample, extracting genomic DNA of viruses and bacteria by virus and bacterial DNA extraction kits, mixing in an equal proportion and taking as a reaction template of LAMP;
(2) forming a loop-mediated isothermal amplification reaction system by mixing the multiple LAMP primer set with a loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, calcein solution containing MnCl2 and genomic DNA of a sample, conducting a loop-mediated isothermal amplification reaction;
wherein a reaction condition for the loop-mediated isothermal amplification reaction is: reaction at 62° C. for 30 minutes, and termination at 80° C. for 5 minutes;
(3) detecting whether the sample contains Vibrio harveyi, Vibrio parahaemolyticus, or Singapore grouper iridovirus by a calcein fluorescence colorimetric method and/or a gel electrophoresis analysis after the amplification reaction is completed.