RPA-LFD primer group and kit for detecting gall midge melansendan and application of RPA-LFD primer group and kit

Abstract

The invention discloses an RPA-LFD (recombinase polymerase amplification-lateral flow dipstick) primer group and a kit for detecting gall midge melansendan and application of the RPA-LFD primer group and the kit, the primer group comprises an Md-AF1-c primer and an Md-AR1-a primer, the sequence of the Md-AF1-c primer is FAM-AACAATAAGTAAAAATCATAT AAAGATAGT, and the sequence of the Md-AR1-a primer is shown in the description. The sequence of the Md-AR1-a primer is Bioton-TTCAGTAGCTCATATAGGAATAGTTTTAGG, and the sequence of the Md-AR1-a primer is shown in the specification. According to the method, the reaction can be completed only through a portable constant temperature device (even body temperature) in a breakthrough mode, the matched freeze-drying reagent can be stored at the room temperature, and the stability and field applicability of the method are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of Hessian gall midge identification technology, specifically relating to an RPA-LFD primer set, reagent kit, and its application for detecting Hessian gall midge. Background Technology

[0002] The Hessian gall midge (Mayetiola destructor (Say)) belongs to the order Diptera, family Cecidomyiidae, subfamily Cecidomyiinae, and genus Mayetiola. It is a major global pest of wheat and a quarantine pest for imported plants. The Hessian gall midge can have 1-6 generations per year in various regions. It overwinters as a third-instar larva, hiding within a pupal ring on winter wheat plants or wheat stubble. The following spring, the mature larvae pupate and emerge as adults. Adults do not feed and have a lifespan of approximately 3 days. Eggs are mostly laid in the vein grooves on the upper surface of the host plant's leaves. After hatching, the larvae crawl along the vein grooves into the leaf sheath and stem to feed and cause damage. Even a single larva can cause stunted plant growth or even death, resulting in reduced yield. Besides wheat, this insect can also feed on and damage barley, rye, *Agrostis* species, *Agrimonia pilosa*, and many other grasses. The Hessian gall midge has a wide suitable habitat, and if it spreads domestically, it will pose a serious threat to wheat production. Therefore, strengthening its quarantine, monitoring, and control is crucial.

[0003] Currently, the identification of *Hemiberlesia echinensis* mainly relies on the morphological characteristics of the adults. The main distinguishing features of the adults are as follows: They resemble small mosquitoes in shape, and are grayish-black. Their body length is 3-4 mm, with females larger than males. The head, compound eyes, and dorsal surface of the thorax are black, while the lateral sides of the thorax and abdomen are yellowish-brown or reddish-brown. The maxillary palps have four segments: the first segment is short, the second is thicker, the third is longer, and the fourth is more than one-third the length of the third segment. The antennae are yellow or yellowish-brown, with 16-18 segments; males typically have 17 segments, and females typically have 16. The coxae are awl-shaped, the pedicels are spherical, and the flagella are conical, with annular fimbriae and erect short hairs. The male antennal flagella have a nearly transparent, slender stalk. The dorsal surface of the thorax has two distinct longitudinal stripes; the scutellum is black with black hairs. The wings are relatively broad, densely covered with short hairs, with longer hairs along the posterior margin. The wing veins are simple; the leading vein (C) is pale brown with a pink base; the first longitudinal vein (R1) is very short, almost merging with the leading vein and connecting to the middle of the leading edge of the wing; the second longitudinal vein (Rs) is more developed and straight, curving slightly downwards near the wingtip and connecting to the trailing edge before the wingtip; the bifurcation of the Cu vein, the junction of the Cu2 vein and the trailing edge, and the end of the R1 vein are almost in a straight line. The halteres are well-developed, pale red, and covered with uneven black scales. The legs are slender, with five tarsal segments, the first being the shortest and the second the longest. Segment V has a pair of curved, slender claws near the end, with an interclaw process between the two claws and a claw pad longer than the claws; both male and female claws are single and toothless. Each abdominal segment has a large square black spot on each side of the tergite. The female ovipositor consists of three segments, cylindrical, pale pink, with the last segment ending in brown. The male's abdomen is pale pink on the last segment, with a pair of brown claspers containing the genitals. The upper genital plate is wider, with numerous sensory pores and deeply recessed notches, mostly V-shaped, and a few slightly U-shaped. The lower genital plate is narrower, with 4-5 nipple-like projections generally visible at the lateral ends. The cerci are two-segmented; the first segment is robust, and the second segment is slender, nearly four times its width in length, and ends in claws.

[0004] Morphological identification methods require a high degree of specimen integrity and the expertise of the identifyer. At ports of entry quarantine, intercepted insects are typically larvae or pupae, making morphological identification difficult. In field monitoring, adult insects obtained through pheromone traps often contain a large number of non-target species, and the captured insects are frequently damaged, affecting the efficiency and accuracy of identification.

[0005] In recent years, molecular detection techniques have been gradually applied to the identification of this insect. Chinese patent application CN114807383A discloses a pair of primers specific to *Hemiberlesia serratifolia*, characterized in that the primer pair includes an upstream primer HFSP-1 and a downstream primer HFSP-2. However, this patent application has problems such as a cumbersome operation process, reliance on laboratory equipment, and long processing time.

[0006] Another patent application, CN118600017A, discloses a LAMP-LFD primer set for detecting black serpentine gall midge, including inner primer FIP / BIP, outer primer F3 / B3, specific probe LF, and loop primer LB (SEQ ID NO: 1-6). However, it still has problems such as easy to produce false positives, strict reagent storage conditions, high reaction temperature, and inconvenience for field or on-site application.

[0007] Therefore, whether for rapid quarantine at ports of entry or real-time monitoring in the field, there is an urgent need to establish a faster, more accurate, stable, and field-applicable molecular detection technology for Black Forest gall midge. Summary of the Invention

[0008] The purpose of this invention is to provide a rapid, accurate, and reliable identification technique for black serpentine gall midges, specifically involving primer sets, kits, and their applications based on recombinase polymerase amplification combined with lateral flow chromatography strip detection (RPA-LFD).

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] 1. RPA-LFD primer set

[0011] This invention provides an RPA-LFD primer set for detecting black serpentine gall midge, comprising the following sequences:

[0012] Upstream primer Md-AF1-c: 5′-FAM-AACAATAAGTAAAAATCATCATAAAGATAGT-3′;

[0013] Downstream primer Md-AR1-a: 5′-Biotin-TTCAGTAGCTCATATAGGAATAGTTTTAGG-3′;

[0014] 2. Test kit

[0015] The present invention further provides a kit for detecting RPA-LFD of black serpentine gall midge, which contains the above-mentioned primer pair.

[0016] In a preferred embodiment of the present invention, the kit further includes: DNA extraction reagent, negative control (enzyme-free water); and lateral flow chromatography strip (LFD).

[0017] 3. Detection performance

[0018] In this invention, the detection limit of agarose gel electrophoresis is 10. -1 ng / μL; the detection limit using the side-flow chromatography test strip is 10 ng / μL. -2 The latter has a sensitivity 10 times that of the former (ng / μL).

[0019] 4. Application Conditions

[0020] The recommended RPA reaction temperature for the kit described in this invention is generally between 37 and 42°C. Currently, numerous studies have shown that the reaction temperature range for RPA-LFD technology is between 30 and 45°C, and the reaction time is generally between 5 and 25 minutes. Outside this temperature range, excessively high or low temperatures will inhibit the reaction efficiency of recombinase and polymerase. In this study, after optimizing the reaction time and temperature, the final reaction conditions were determined to be a reaction at an arbitrary constant temperature of 32–40°C for 18 minutes. Under these conditions, the bands of the amplified products appear clearer and brighter in agarose gel electrophoresis, and the detection time is effectively saved, improving detection efficiency. In practical applications, it can also be combined with the toothpick method for DNA extraction from the sample to simplify the pretreatment steps.

[0021] 5. Technological Advantages and Features

[0022] This invention establishes a rapid detection technology system for *Hemiberlesia serrata* RPA-LFD based on the mitochondrial ND4 gene sequence. Amplification can be completed within 18 minutes under isothermal conditions within the range of 32–40 °C, with a detection limit of 10. -2 ng / μL.

[0023] This method has the following key features:

[0024] (1) Unique target gene: Unlike most animals, primers are designed based on the mitochondrial COI sequence. Through systematic screening, this invention has obtained highly specific RPA primers from the mitochondrial ND4 gene of the black gall midge for the first time, which confirms the value of this gene in the identification of the black gall midge.

[0025] (2) Low equipment requirements: Only a portable thermostat is needed, and the reaction can even be driven by body temperature;

[0026] (3) Good reagent stability: The matching lyophilized reagent can be stored at room temperature, which is convenient for transportation and has strong stability and on-site applicability;

[0027] (4) Strong compatibility with pretreatment: It is well compatible with the toothpick method for DNA extraction, and only 23 minutes are needed from sample processing to result interpretation;

[0028] (5) Wide range of insect stages: It can accurately detect the larvae, pupae and adults of the black forest gall midge after 5 days of exposure, which can meet the multiple requirements of port quarantine and field monitoring for rapid, portable and accurate detection. Attached Figure Description

[0029] Figure 1Results of amplification efficiency test of the RPA-LFD primer set for *Hemiberlesia serratifolia*, where M: 2000 bp DNA Marker, N: enzyme-free water; 1~5: represent different primer sets of Md-RPA-LFD, respectively.

[0030] Figure 2 RPA-LFD specificity test results, including a. agarose gel electrophoresis results, b. lateral flow chromatography (LFD) results; M: 2000 bp DNA Marker, N: enzyme-free water; Md: *Gallus gallus domesticus*, Bo: *Amanita muscaria*, Sm: *Sinomenis rubrum*, Ed: *Sinomenis camouflageus*, Et: *Sinomenis rubrum*.

[0031] Figure 3 The reaction time test results of the RPA-LFD system under constant temperature of 37.0 ℃, including a. agarose gel electrophoresis detection results, b. side-flow chromatography test strip detection results; M: 2000 bp DNA Marker; 1-8: 12, 14, 16, 18, 20, 22, 24, 26 min;

[0032] Figure 4 Results of RPA-LFD reaction at different temperatures after 18 min, including a. agarose gel electrophoresis results, b. side-flow chromatography test strip results; M: 2000 bp DNA Marker; 1~9: 24, 26, 28, 30, 32, 34, 36, 38, 40 ℃;

[0033] Figure 5 RPA-LFD sensitivity test results, including a. agarose gel electrophoresis results, b. side-flow chromatography test strip results; M: 2000 bp DNA Marker; 1-6: DNA concentrations of 10 -4 10 -3 10 -2 10 -1 1, 10 ng / μL;

[0034] Figure 6 The results of RPA-LFD field sample validation for *Hemiberlesia echinensis* are shown in the following figures: a. Agar gel electrophoresis results; b. Lateral flow chromatography strip results; M: 2000 bp DNA Marker; N: Enzyme-free water; 1: Larva; 2: Pupa; 3: Adults exposed to traps for 5 days; Repeated 3 times. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1:

[0037] 1. Materials and Methods

[0038] 1.1 Test insect source

[0039] Black serpentine gall midge (original population from Wenquan County, China) was bred at the Yining Customs Technology Center (21 ℃, RH 75%, L:D = 16:8). Control insects included: *Elachiptera tuberculifera* (larvae and pupae collected from wheat fields in Bole City and Chabuchar County in June 2023 and 2024), *Elachiptera tuberculifera* (adults collected from wheat fields in Bole City in 2023 using a net-sweeping method), *Bradysia odoriphaga* (adults obtained during the rearing of *Black serpentine gall midge*), and *Sitodiplosis mosellana* (larvae provided by Professor Cheng Weining of the College of Plant Protection, Northwest A&F University). All collected samples were preserved by immersion in 100% ethanol at -80 ℃.

[0040] 1.2 Main Reagents

[0041] The basic nucleic acid amplification kit (RPAID method) and lateral flow chromatography test strips were purchased from Lesun Biotechnology (Wuxi) Co., Ltd.; DL2000 bp DNA Marker, gel electrophoresis loading buffer (6× Loading buffer) and agar powder were purchased from Hunan Aikerui Biotechnology Co., Ltd.; the blood / cell / tissue genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd.; and enzyme-free water was purchased from Beijing Solarbio Technology Co., Ltd.

[0042] 2 Primer Design and Screening

[0043] RPA-LFD primers need to meet the following conditions: (1) Primer length is 28-35 bp; (2) High base randomness, GC content is between 30-70%, consecutive Gs are avoided at the 5' end of the primer, and the 3' end ends with G or C; (3) Wide annealing temperature to avoid consecutive base repetitions, palindromic structures and primer dimers; (4) Amplified fragment length is 150-300 bp; (5) Primers must be designed based on suitable specific sites and experimentally screened. Given that long-fragment primer amplification is prone to dimers and hairpin structures and there is currently no primer design evaluation software suitable for RPA amplification, RPA primer and probe screening and system optimization are crucial.

[0044] DNA target sequence selection is crucial for specific primer design. Molecular identification of most insect species relies on the relatively conserved nature of mitochondrial cytochrome oxidase (COI) gene sequences to screen for specific primers. However, the inventors designed 110 primer sets from the mitochondrial COI sequence of *Gallus niger*, but after testing, all showed poor specificity or produced palindromic structures and primer dimers, failing to meet the detection requirements of *Gallus niger* RPA-LFD technology. Subsequent extensive analysis and comparison of conserved regions of *Gallus niger* mitochondrial DNA revealed that the NADH dehydrogenase 4 gene (ND4) exhibits intraspecific conservatism and rich interspecific variation, meeting the criteria for screening specific primers. Therefore, ND4 was used as the target for primer design, and 5 sets were initially selected from 38 primer sets for specificity verification. Primer synthesis was commissioned to General Biotechnology (Anhui) Technology Co., Ltd. (Table 1).

[0045] Table 1. Information on the RPA-LFD primer set for *Hessian gall midge*.

[0046]

[0047] 3. DNA extraction

[0048] Insect DNA was extracted using the Tiangen Animal Tissue DNA Kit for subsequent primer screening, reaction system optimization, and sensitivity testing. DNA was rapidly extracted from field samples using the toothpick method to evaluate the practicality of the RPA-LFD detection technology system.

[0049] 4. RPA-LFD reaction system and operation method

[0050] The total volume of the RPA-LFD reaction system was 47 μL (Table 2).

[0051] Table 2 RPA-LFD reaction system and dosage

[0052]

[0053] Transfer the above 47 μL premixed solution to the reaction unit, mix manually and then centrifuge briefly (4000 rpm, 5 s). Then add 3 μL of initiator to the inner wall of the reaction tube cap, tighten the cap and centrifuge briefly (4000 rpm, 5 s) to mix the reagents, mix by hand and centrifuge briefly again (4000 rpm, 5 s), and immediately place the reaction tube in a 37 ℃ constant temperature metal bath for 25 min. The reaction products are detected by the following two methods: (1) 1.5% agarose gel electrophoresis. Take 20 μL of the reaction product and mix it with an equal volume of phenol chloroform solution (phenol: chloroform: isoamyl alcohol = 25:24:1), centrifuge at 12,000 rpm for 2 min and take 5 μL of supernatant, add 3 μL of loading buffer and mix, and then perform electrophoresis analysis. If a specific band appears, it is a positive reaction; if no band appears, it is a negative result. (2) Lateral flow chromatography. Dilute 10 μL of the product 20 times with sterile water and insert it into the lateral flow test strip. If the test line (T) turns red and the control line (C) turns blue within 3 minutes, the result is positive. If only the control line turns blue and the test line does not appear, the result is negative.

[0054] The initial reaction conditions were a constant temperature reaction of 37 °C for 25 min (Takara PCR amplification instrument).

[0055] 5. Primer amplification efficiency analysis

[0056] The amplification efficiency of each primer set for each RPA-LFD was evaluated by 1.5% agarose gel electrophoresis. Observation was performed in a gel electrophoresis imaging system.

[0057] The RPA-LFD primer set of *Gnaphalium affine* with clear amplification strips and no nonspecific products was screened and modified at the 5' end. The 5' end of the forward primer (AF) was modified with carboxyfluorescein (FAM), and the 5' end of the reverse primer (AR) was modified with biotin.

[0058] 6. Primer specificity verification

[0059] Using *Hemiberlesia euphratica* DNA as a positive control, DNA from four non-target insect species as negative controls, and enzyme-free water as a blank control, three replicates were performed. The reaction system and procedure are shown in Table 2. Primer specificity was evaluated by 1.5% agarose gel electrophoresis and lateral flow test strip detection, respectively.

[0060] 7. Optimization of reaction conditions

[0061] Using *Gallus niger* DNA as a template, the reaction conditions were systematically optimized using the controlled variable method. The effects of reaction time (12-26 min) and temperature (24-40 ℃) on amplification efficiency were investigated to determine the optimal reaction parameters. The reaction time gradients were set to 12, 14, 16, 18, 22, 24, and 26 min, and the reaction temperature gradients were set to 24, 26, 28, 30, 32, 34, 36, 38, and 40 ℃.

[0062] 8. Sensitivity Test

[0063] The DNA of *Gallus niger* was serially diluted (10–10⁻⁻⁶). 4 2 μL of DNA dilution buffer (ng / μL) was used for RPA-LFD amplification. The optimized reaction system from step 7 was used for amplification. The detection limit was determined by 1.5% agarose gel electrophoresis and lateral flow test strip detection, with enzyme-free water as a blank control.

[0064] 9. Simulated field effect test

[0065] Three larvae, three pupae, and three adults of the black sedge were collected from the field. DNA was extracted using the toothpick method and then analyzed by RPA-LFD to evaluate the applicability of this method under field conditions. The RPA-LFD system is shown in Table 2, and the reaction parameters were the optimized reaction conditions described in Section 7. The RPA-LFD products were detected by 1.5% agarose gel electrophoresis and lateral flow test strips. Enzyme-free water was used as a blank control, and the experiment was repeated three times.

[0066] 10 Results and Analysis

[0067] 10.1 Primer amplification efficiency analysis

[0068] Gel electrophoresis results showed that the first group of RPA primers had the highest amplification efficiency, producing clear and bright specific bands, and the amplification products were pure with no interference from other bands. Figure 1 ), which was selected as the standard primer set for subsequent experiments.

[0069] The 5' ends of the upstream primer (AF) and downstream primer (AR) of the selected black serpentine gall midge RPA-LFD primers were modified with FAM and Biotion, respectively (Table 3).

[0070] Table 3. Information on the modified RPA-LFD primer set for *Gallus niger*.

[0071]

[0072] 10.2 Primer Specificity Verification

[0073] Agarose gel electrophoresis results showed that only the *Hemiberlesia serratifolia* DNA sample showed a clear and bright target band, while no amplification products were found in the control insect DNA samples. Figure 2 a). The test strip results showed that only the detection line (T) and control line (C) of the *Gnaphalium affine* DNA sample turned red and blue, respectively, while only the control line (C) of the control samples turned blue. Figure 2 b). This indicates that the primers have good specificity.

[0074] 10.3 Optimization of the reaction system

[0075] Electrophoresis results showed that the Md-RPA-LFD system began to show clear bands after 16 min of reaction at a constant temperature of 37 ℃, and the bands appearing in the range of 18-26 min were clearer and brighter. Figure 3 a); The test strip results showed that at 16 minutes, the test line and the control line were red and blue, respectively. Figure 3 (b) indicates that amplification can be detected after reacting at 37 ℃ for 16 min, and the results are clearer after 18~26 min. Considering the accuracy and stability of the detection results, 18 min is determined to be the optimal detection time.

[0076] Electrophoresis results showed that the Md-RPA-LFD reaction system could produce bright electrophoretic bands after 18 min under any isothermal conditions within the range of 32~40 ℃. Figure 4 a) The results of the lateral flow test strips showed that both the detection line and the control line appeared within the range of 30~40 ℃. Figure 4 (b) indicates that the suitable temperature range is 30~40 ℃. To ensure the stability and accuracy of the results, the optimal conditions are recommended to be 32 ℃ for 18 min.

[0077] 10.4 Sensitivity Test

[0078] Electrophoresis results showed that when the sample DNA concentration was ≥10... -1 At ng / μL, clear amplification bands can be observed ( Figure 5 a) The test strip results showed that the sample DNA concentration was ≥10. -2 At ng / μL, the detection line appears distinctly red ( Figure 5 b). This indicates that the detection limit of electrophoresis is 10. -1 The detection limit for the test strip method is 10 ng / μL. -2 The latter has a sensitivity 10 times that of the former (ng / μL).

[0079] 10.5 Field Sample Validation

[0080] DNA from all life stages of *Gnaphalium affine* extracted using the toothpick method was positive for Md-RPA-LFD testing. Figure 6 This indicates that the method is suitable for rapid field detection.

[0081] 11 Summary

[0082] This invention establishes an RPA-LFD detection method based on the ND4 gene of the black serpentine gall midge. Amplification can be completed within 18 minutes at 32-40 °C, with a minimum detectable sample DNA concentration of 10. -2 ng / μL. Considering temperature fluctuations during field applications, the recommended reaction conditions are 32–40 ℃ for 18 min to ensure accuracy and stability of results. Combined with toothpick-based DNA extraction, the entire process from sample to result can be completed within 23 min. This method is simple to operate, provides stable results, and is suitable for field applications, meeting the needs of port recommendations and field monitoring for rapid identification of *Gallus niger*. During this invention, *Gallus niger*-specific RPA primers could not be screened from the COI gene sequence in mitochondrial genes, but specific primers were screened from the ND4 gene, indicating that the ND4 gene can also serve as a novel molecular target sequence for species identification.

[0083] The preferred embodiments of the present invention have been described in detail above. However, the present disclosure is not limited to the specific details of the above embodiments. Any equivalent substitutions or improvements made without departing from the concept of the present invention should be included within the protection scope of the present invention.

Claims

1. An RPA-LFD primer set for detecting *Gallus niger*, comprising Md-AF1-c and Md-AR1-a primers, wherein, The sequence of the Md-AF1-c primer is FAM-AACAATAAGTAAAAATCATCATAAAGATAGT; The sequence of the Md-AR1-a primer is Biotion-TTCAGTAGCTCATATAGGAATAGTTTTAGG.

2. A kit for detecting RPA-LFD in *Gallus niger*, the kit comprising Md-AF1-c and Md-AR1-a primers, wherein, The sequence of the Md-AF1-c primer is FAM-AACAATAAGTAAAAATCATCATAAAGATAGT; The sequence of the Md-AR1-a primer is Biotion-TTCAGTAGCTCATATAGGAATAGTTTTAGG.

3. The kit according to claim 2, wherein the kit comprises DNA extraction reagent.

4. The kit according to claim 2, wherein the kit comprises enzyme-free water as a control.

5. The kit according to any one of claims 2-4, wherein the kit is a lateral test strip.

6. The use of the kit according to any one of claims 2-5 in the detection of Hessian gall midge.

7. According to claim 6, the primers in the kit can complete the reaction in just 18 minutes under any constant temperature condition of 32~40 °C.

8. The application according to claim 6, further comprising extracting DNA from the sample to be tested using a toothpick DNA extraction method.

Images