KR-102963583-B1 - A hydrolysis probe-based primer composition for detection of Coptotermes gestroi and diagnostic kit comprising the same

Abstract

The present invention relates to a hydrolysis probe-based primer composition for identifying Asian underground termites (tentative name, Coptotermes gestroi ) and a diagnostic kit containing the same, and more specifically, to a hydrolysis probe-based primer composition for identifying Asian underground termites ( Coptotermes gestroi ) and a diagnostic kit containing the same that can rapidly and accurately identify Asian underground termites from domestic and foreign termite species.

Inventors

• 박영준
• 조영호
• 채도영

Assignees

• 국립생태원

Dates

Publication Date

20260512

Application Date

20240621

Claims (5)

1. A hydrolysis probe-based primer composition for identifying Asian underground termites ( Coptotermes gestroi ), comprising a forward primer of SEQ ID NO. 2, a reverse primer of SEQ ID NO. 7, and a probe of SEQ ID NO. 12, wherein A hydrolysis probe-based primer composition for identifying Coptotermes gestroi, characterized in that the above primer composition is used to distinguish Coptotermes gestroi from termites including Reticulitermes kanmonensis , Reticulitermes speratus , Cryptotermes domesticus , and Coptotermes gestroi .
2. A diagnostic kit for identifying Asian underground termites ( Coptotermes gestroi ) comprising a hydrolysis probe-based primer composition for identifying Asian underground termites ( Coptotermes gestroi ) according to claim 1, wherein A diagnostic kit for identifying Asian subterranean termites ( Coptotermes gestroi ), characterized by being used to distinguish Asian subterranean termites ( Coptotermes gestroi ) from termites including Kanmon termites ( Reticulitermes kanmonensis ), termites ( Reticulitermes speratus ), dry wood termites ( Cryptortes domesticus ), and Asian subterranean termites ( Coptotermes gestroi ).
3. Step of extracting DNA from a sample; The step of mixing the above-mentioned extracted DNA; and the hydrolysis probe-based primer composition for identifying Asian underground termite ( Coptotermes gestroi ) according to claim 1, and amplifying by PCR; and A method for identifying Asian underground termites ( Coptotermes gestroi ), comprising the step of verifying the CT (threshold cycle) value of the amplified product, A method for identifying Coptotermes gestroi, characterized by being used to distinguish Coptotermes gestroi from termites including Reticulitermes kanmonensis , Reticulitermes speratus , Cryptotermes domesticus , and Coptotermes gestroi .
4. In paragraph 3, A method for identifying Asian underground termites ( Coptotermes gestroi ) characterized by the fact that an amplification curve is observed specifically only in Asian underground termites ( Coptotermes gestroi ) during a PCR amplification reaction.
5. In paragraph 4, A method for identifying Asian underground termites ( Coptotermes gestroi ), characterized by the step of checking the CT value of the amplified product above, determining it as Asian underground termite ( Coptotermes gestroi ) when CT appears at a maximum number of cycles or less.

Description

A hydrolysis probe-based primer composition for detection of Asian underground termite (Coptotermes gestroi) and diagnostic kit comprising the same The present invention relates to a hydrolysis probe-based primer composition for identifying Asian underground termites (tentative name, Coptotermes gestroi ) and a diagnostic kit containing the same, and more specifically, to a hydrolysis probe-based primer composition for identifying Asian underground termites ( Coptotermes gestroi ) and a diagnostic kit containing the same that can rapidly and accurately identify Asian underground termites from domestic and foreign termite species. Termites are insects belonging to the family Rhinotermitidae of the order Blattodea, and currently about 2,972 species have been described. Termites are classified as pests because they feed on wood. Termites that live underground enter the interior of wood through wooden structures in contact with the ground. They inhabit any region with an average annual temperature of 10°C or higher and have a habit of consuming the interior of wood to hollow it out, then spreading to nearby wood. Therefore, in the case of wooden structures such as cultural heritage, the lower part of the pillars supports the load, so damage caused by termites can lead to the collapse of the building itself. There are a total of three termite species inhabiting Korea. The termite formally reported to the academic community ( *Reticulitermes speratus kyushuensis *) is known to be distributed nationwide, including Jeju Island, while the Kanmon termite ( *Reticulitermes kanmonensis *) is distributed in the Chungcheong and Jeolla regions along the Geum River basin. The whole termite ( *Glyptotermes nakajimai *) is reported to inhabit only Yeoseodo Island in Wando-gun, Jeollanam-do. These three species have consistently established themselves as natives in Korea, and their severity is reported to be lower compared to invasive species. According to reference materials on preventing damage from high-risk termites in Hanok construction distributed by the Architectural Policy Bureau of the Ministry of Land, Infrastructure and Transport in 2014, the probability of two species of high-risk termites ( Coptotermes formosanus and Cryptotermes domesticus ), known to cause the most damage worldwide, entering the country is very high, and it was reported that if they enter the country under climate change scenarios, they could become established in the future and the damage would be at a 'disaster level'. According to 2014 data, a total of 63 cases of termites were detected in plant quarantine over the past 15 years, and 72% of them were identified as high-risk termites. According to data released by the Ministry of Agriculture, Food and Rural Affairs in 2023, termites were detected 30 times during the import quarantine process over the 10 years from 2013 to 2023. According to data published by the Biodiversity Division of the Ministry of Environment on September 26, 2023, an alien termite was detected in Changwon, and species analysis revealed it to be the Western drywood termite (tentative name, Incisitermes minor ), and in May 2023, the drywood termite ( Cryptotermes domesticus ) was detected in a house in Gangnam-gu, Seoul. Currently, research on termite genomes is very lacking both domestically and internationally, and there are no commercially available diagnostic kits for species identification, nor is it difficult to identify species or subspecies based on COI. Therefore, it is necessary to develop technology capable of rapidly and accurately identifying Asian underground termites from domestic and foreign termite species. Figure 1 shows the melting curve and electrophoresis results during DNA amplification of four species of termites using primer set 1 (B2_Cop.gestroi_Primer1) of the present invention. Figure 2 shows the melting curves and electrophoresis results during DNA amplification of four species of termites using primer set 2 (B2_Cop.gestroi_Primer2) of the present invention. Figure 3 shows the melting curve and electrophoresis results during DNA amplification of four species of termites using primer set 3 (B2_Cop.gestroi_Primer3) of the present invention. Figure 4 shows the melting curve and electrophoresis results during DNA amplification of four species of termites using primer set 4 (B2_Cop.gestroi_Primer4) of the present invention. Figure 5 shows the melting curve and electrophoresis results during DNA amplification of four species of termites using primer set 5 (B2_Cop.gestroi_Primer5) of the present invention. Figures 6 to 8 show the amplification curves of the real-time PCR amplification reaction of four types of termites of the present invention. The present invention will be described in detail below based on the embodiments and drawings. The terms, embodiments, drawings, etc. used in this invention are merely illustrative to explain the invention more specifically and to aid the understanding of those