US-12618116-B2 - Specific primers for identifying Asian gypsy moth and method of detection thereby

US12618116B2US 12618116 B2US12618116 B2US 12618116B2US-12618116-B2

Abstract

Disclosed is a pair of specific primers for identifying Asian gypsy moth and a method of detection thereby. In the present disclosure, based on the SS-COI technology, primers for distinguishing other species of Lymantria are designed according to the specific region of the mitochondrial COI gene of Asian gypsy moth. The pair of primers has accurate identification effects on different geographical populations, different insect states and different instars of Asian gypsy moth; in addition, the whole detection process only takes 2-3 hours, which has the advantages of rapidity and accuracy.

Inventors

Juan Shi
Wenzhuai JI
Shuyue LIU

Assignees

BEIJING FORESTRY UNIVERSITY

Dates

Publication Date: 20260505
Application Date: 20220915
Priority Date: 20211224

Claims (2)

1 . A method for detecting Asian gypsy moth, comprising the following steps: (1) extracting genomic DNA of an insect to be tested; (2) performing PCR amplification using a pair of specific primers and genomic DNA as a template, wherein the specific primers comprise a forward primer consisting of SEQ ID NO: 1 and a reverse primer consisting of SEQ ID NO:2 and PCR amplification reaction comprises 12.5 μl of 2×PCR premix, 1-2 μl of 10 pmol/μl forward and reverse primers, 1-3 μl of DNA template with a concentration of 20 ng/μl, with a total volume of 25 μl; wherein the PCR amplification conditions comprise an initial denaturation for 2 min at 94° C.; followed by 30 cycles of denaturation at 94° C. for 30 s, annealing at 49° C. to 56° C. for 30 s, and extension at 72° C. for 1 min; with a final extension at 72° C. for 5 min; and resulting products are stored at 40° C.; wherein the PCR premix comprises a Taq DNA polymerase, a deoxyribonucleoside triphosphate (dNTP), a magnesium ion, and a reaction buffer; and (3) analyzing amplified products; wherein analyzing is performed by detecting the amplified products by agarose gel electrophoresis, and if a characteristic band of 350 bp appears, determining that the insect detected is Asian gypsy moth.
2 . The method according to claim 1 , wherein the temperature for the annealing is 50° C.

Description

CROSS REFERENCE TO RELATED APPLICATION This patent application claims the benefit and priority of Chinese Patent Application No. 202111593718.2, entitled “Specific primers for identifying Asian gypsy moth and method of detection thereby” filed on Dec. 24, 2021, the disclosure of which is incorporated by reference herein in its entirety as part of the present application. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The xml file titled “HLP20220400312-Sequence_Listing” created Sep. 5, 2022 and being 8.27 KB in size, as filed with the present application via the USPTO patent electronic filing system, is incorporated by reference herein in its entirety as part of the present application. The xml file titled “HLP20220400312-Sequence Listing rev” created Oct. 21, 2022 and being 4.30 KB in size filed via the USPTO patent electronic filing system on Oct. 28, 2022 is incorporated by reference herein in its entirety as part of the present application. TECHNICAL FIELD The present disclosure relates to the field of forestry and plant quarantine, and in particular to specific primers for identifying Asian gypsy moth and a method of detection thereby. BACKGROUND ART The gyspy moth (spongy moth) Lymantria dispar Linnaeus is a widely distributed leaf-eating pest. According to the morphological characteristics, flight ability of females and geographical distribution, the latest subspecies classification research has divided them into three typical subspecies (Pogue& Schaefer, 2007): Lymantria dispar dispar, Lymantria dispar asiatica and Lymantria dispar japonica. The Lymantria dispar dispar is also known as European gypsy moth, Lymantria dispar asiatica and Lymantria dispar japonica are collectively referred to as Asian gypsy moth. Compared with the European gypsy moth, the female adult of the Asian gypsy moth has strong flight ability and its larvae have a wide range of hosts, with more than 500 kinds of forest trees serving as its host, while the female adult of the European gypsy moth has no flight ability and has about 250 species of larva hosts. Due to the migration distance of Asian female adults up to 25-40 miles and the characteristics of their wide host range, once introduced and established, Asian gypsy moth will speed much faster than European gypsy moth. Their harm to North America will be more serious than that of European gypsy moth. In order to prevent the introduction of Asian gypsy moth, the United States has successively imposed special quarantine measures to countries and regions where AGM is distributed. The establishment of quarantine measures has played an effective role in preventing the introduction of Asian gypsy moths. It can be seen that the Asian gypsy moth causes serious effects on the trade between countries, and also has a potential threat to the ecological environment. The destructive power and quarantine status of the Asian gypsy moth have greatly affected the phytosanitary and foreign friendly trade at important ports in China, which is the birthplace of the Asian gypsy moth. However, there are still several kinds of tussock moths, such as Lymantria xylina, Lymantria apicebrunnea, Lymantria monacha and Lymantria mathura. The morphology of their adults, larvae and even eggs is so similar to those of the Lymantria dispar that even the professional can also make false identifications. Because the species of Lymantria are mainly identified based on the morphology of lepidotic wing, and the moths found during the transportation of cargos are rarely with complete lepidotic wing pattern. In the process of port quarantine and cargo transportation, the traditional morphological methods can not meet the practical needs of rapid and accurate recognition and identification of Lymantria dispar and its related species. Therefore, it is very likely that other non-quarantine species of Lymantria will be identified as Lymantria dispar during quarantine, which may lead to the wrong direction for the establishment of quarantine measures. Therefore, rapid identification and accurate recognition of different species of Lymantria are of great significance for the implementation of control policies. Given the quarantine significance of Lymantria dispar and the similar morphological characteristics of different species of Lymantria, in recent years, many studies have used molecular biology methods to develop rapid detection primers between Asian gypsy moth and European gypsy moth and other species of Lymantria, in which mitochondrial DNA has been proved to be a good target for rapid identification. Mitochondrial DNA (mtDNA) is characterized by simple structure, fast mutation rate (usually 5-10 times that of single-copy nuclear DNA), strong gene conservation, and matrilineal inheritance. It is called the second genetic information system of eukaryotic cells, or the extranuclear expression system. Compared with the nuclear genes, mtDNA can better reflect the evolutionary events in a short period of time, especial