Visual detection kit for rapidly identifying book lice, detection method and application of visual detection kit

Abstract

The invention discloses a visual detection kit for rapidly identifying book lice, a detection method and application of the visual detection kit. On the basis of a multi-enzyme constant-temperature rapid amplification technology, two specific primers and probes for identifying the booklice are designed, and visual detection of the booklice is realized through a lateral flow chromatography paper strip. The method for rapidly identifying the booklice established on the basis of a multi-enzyme constant-temperature rapid amplification technology and a lateral flow chromatography test strip technology can be used for rapidly identifying eggs, nymphs and adults of the booklice in combination with a booklice DNA (Deoxyribonucleic Acid) crude extraction technology, so that the dependence on instruments and equipment in the identification process is avoided; and the kit also has the characteristics of rapidness, high efficiency, strong specificity, high sensitivity, convenience in operation, no influence of environmental conditions and the like, is suitable for on-site rapid detection, and is easy to popularize in grass-roots. The invention provides a reliable technical basis for prevention and control of the book lice, and has important significance for grain storage protection, prevention and control of alien invasive organisms, assistance of international trade and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a visual detection kit for rapidly identifying Liposcelis entomophila, a detection method and its application, and more particularly to a visual detection kit for rapidly identifying Liposcelis entomophila based on multi-enzyme isothermal rapid amplification technology combined with lateral flow chromatography strip technology, a detection method and its application. Background Art

[0002] Booklice belong to the genus Liposcelis of the family Liposcelididae in the order Psocoptera, and are distributed globally. They are a common storage pest. Booklice are tiny (0.6 - 1.5 mm), mainly causing damage by group feeding, and are also prone to causing the heating and mildew of stored grains, posing a threat to food safety and grain trade. Currently, a total of 127 species of insects in the genus Liposcelis have been recorded in the world, and 27 species have been recorded in China. Liposcelis corrodens is one of the ten common storage booklice, mainly distributed in Europe, Oceania and North America, and the countries where it is distributed include the United States, Canada, France, etc. In order to prevent its further spread, it is necessary to continuously improve the identification technology for Liposcelis corrodens.

[0003] Currently, the identification technologies for the genus Liposcelis mainly fall into two categories: morphological identification technology and molecular identification technology. Insects in the genus Liposcelis are small and have similar interspecific morphological characteristics, so morphological identification is somewhat difficult. In terms of molecular identification, traditional molecular identification technologies based on variable-temperature amplification technology, such as DNA barcoding and gene chips, have problems such as being restricted by instruments and long reaction times when applied. The LAMP technology that has been applied in the identification of storage booklice has application defects such as a relatively high reaction temperature, complex primer design, long reaction time, and serious false-positive problems. In order to detect and identify Liposcelis corrodens as early as possible during trade and food storage and prevent its further spread, it is very necessary to develop a simpler, faster and environmentally condition-independent quarantine identification technology.

[0004] Multienzyme Isothermal Rapid Amplification (MIRA) is a constant-temperature nucleic acid rapid amplification system containing multiple enzymes, which uses recombinase, polymerase, etc. for amplification reactions, greatly shortening the DNA amplification time, and can achieve amplification in as short as 5 minutes. In recent years, it has been widely applied in many fields. However, in the identification of storage booklice, especially for the rapid detection of Liposcelis corrodens, no relevant application reports have been seen. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to achieve rapid and accurate identification of Liposcelis corrodens.

[0006] To solve the above technical problems, the present invention first provides a kit of reagents for identifying or assisting in the identification of Liposcelis entomophila.

[0007] The kit of reagents for identifying or assisting in the identification of Liposcelis entomophila provided by the present invention includes primer pairs for identifying or assisting in the identification of Liposcelis entomophila; the primer pairs for identifying or assisting in the identification of Liposcelis entomophila are composed of primer - F and primer - R;

[0008] The primer - F is as follows a1) or a2):

[0009] a1) A single - stranded DNA molecule as shown in Sequence 1 in the sequence listing;

[0010] a2) A single - stranded DNA molecule obtained by substituting and / or deleting and / or adding one or several nucleotides to Sequence 1 and having the same function as Sequence 1;

[0011] The primer - R is as follows a3) or a4):

[0012] a3) A single - stranded DNA molecule as shown in Sequence 2 in the sequence listing;

[0013] a4) A single - stranded DNA molecule obtained by substituting and / or deleting and / or adding one or several nucleotides to Sequence 2 and having the same function as Sequence 2;

[0014] The above - mentioned kit of reagents further includes a probe for identifying or assisting in the identification of Liposcelis entomophila; the probe is composed of a single - stranded DNA molecule shown in Sequence 3, T - tetrahydrofuran, and a single - stranded DNA molecule shown in Sequence 4.

[0015] Furthermore, the 5' - end of the primer - R is labeled with biotin.

[0016] The 5' - end of the probe is labeled with the fluorescent group FAM, and the 3' - end is modified with C3 - Spacer.

[0017] Moreover, the molar ratio of the primer - F, the primer - R, and the probe is 10:10:3.

[0018] To solve the above technical problems, the present invention also provides a new use of the above - mentioned kit of reagents.

[0019] The present invention provides the application of the above - mentioned kit of reagents in any one of the following b1) - b6):

[0020] b1) Identifying or assisting in the identification of whether a to - be - tested booklice is Liposcelis entomophila;

[0021] b2) Preparing a product for identifying or assisting in the identification of whether a to - be - tested booklice is Liposcelis entomophila;

[0022] b3) Detecting or assisting in the detection of whether a to - be - tested sample contains Liposcelis entomophila;

[0023] b4) To prepare a product for detecting or assisting in detecting whether a sample to be tested contains Liposcelis bostrychophila;

[0024] b5) To distinguish or assist in distinguishing Liposcelis bostrychophila from other booklice;

[0025] b6) To prepare a product for distinguishing or assisting in distinguishing Liposcelis bostrychophila from other booklice.

[0026] To solve the above technical problems, the present invention also provides a kit containing the above-mentioned set of reagents;

[0027] The functions of the kit are any one of the following c1)-c3):

[0028] c1) To identify or assist in identifying whether a booklouse to be tested is Liposcelis bostrychophila;

[0029] c2) To detect or assist in detecting whether a sample to be tested contains Liposcelis bostrychophila;

[0030] c3) To distinguish or assist in distinguishing Liposcelis bostrychophila from other booklice.

[0031] Furthermore, the kit further includes a multi-enzyme isothermal rapid amplification reagent and a lateral flow chromatographic test strip.

[0032] Still further, the multi-enzyme isothermal rapid amplification reagent may include enzyme powder freeze-dried particles (reaction tubes containing enzyme powder freeze-dried particles), reaction buffer A buffer, and amplification reaction initiator B buffer.

[0033] Even further, the kit further includes a negative control (such as sterile ultrapure water) and a positive control (such as genomic DNA of Liposcelis bostrychophila), and a lateral flow chromatographic test strip for observing the amplification result.

[0034] In a specific embodiment of the present invention, the enzyme powder freeze-dried particles (reaction tubes containing enzyme powder freeze-dried particles), reaction buffer A buffer, and amplification reaction initiator B buffer all come from a colloidal gold test strip type DNA isothermal rapid amplification kit. The colloidal gold test strip type DNA isothermal rapid amplification kit is a product of Weifang Anpu Future Biotechnology Co., Ltd., with the product number WLN8203KIT. The lateral flow chromatographic test strip is a product of Weifang Anpu Future Biotechnology Co., Ltd., named HybriDetect test strip, with the product number WLFS8201.

[0035] The preparation method of the above kit also belongs to the protection scope of the present invention.

[0036] The preparation method of the kit may be the following d1) or d2):

[0037] d1) Individually package the primer pair and the probe in the above-mentioned kit of reagents;

[0038] d2) Mix the primer pair and the probe in the above-mentioned kit of reagents in a paired manner according to a ratio.

[0039] In d2), mix primer-F and primer-R in the above-mentioned kit of reagents at a molar ratio of 1:1 respectively.

[0040] In d2), mix primer-F, primer-R and the probe in the above-mentioned kit of reagents at a molar ratio of 10:10:3 respectively.

[0041] To solve the above technical problems, the present invention also provides a method for identifying or assisting in identifying whether a to-be-detected booklouse is Liposcelis entomophila.

[0042] The method for identifying or assisting in identifying whether a to-be-detected booklouse is Liposcelis entomophila provided by the present invention includes the following steps: Extract the nucleic acid of the to-be-detected booklouse, use the nucleic acid of the to-be-detected booklouse as a template, and perform multi-enzyme isothermal rapid amplification using the above-mentioned primer pair and probe to obtain a multi-enzyme isothermal rapid amplification product; Dilute the multi-enzyme isothermal rapid amplification product and then add it dropwise to the sample application hole of a lateral flow chromatographic test strip, and determine whether the to-be-detected booklouse is Liposcelis entomophila by observing whether bands appear on the quality control line and the test line; If bands appear on both the quality control line and the test line of the lateral flow chromatographic test strip, the to-be-detected booklouse is or is a candidate to be Liposcelis entomophila; If a band appears on the quality control line of the lateral flow chromatographic test strip and no band appears on the test line, the to-be-detected booklouse is not or is a candidate not to be Liposcelis entomophila.

[0043] To solve the above technical problems, the present invention also provides a method for detecting or assisting in detecting whether a to-be-detected sample contains Liposcelis entomophila.

[0044] The method for detecting or assisting in detecting whether a to-be-detected sample contains Liposcelis entomophila provided by the present invention includes the following steps: Extract the nucleic acid of the to-be-detected sample, use the nucleic acid of the to-be-detected sample as a template, and perform multi-enzyme isothermal rapid amplification using the above-mentioned primer pair and probe to obtain a multi-enzyme isothermal rapid amplification product; Dilute the multi-enzyme isothermal rapid amplification product and then add it dropwise to the sample application hole of a lateral flow chromatographic test strip, and determine whether the to-be-detected sample contains Liposcelis entomophila by observing whether bands appear on the quality control line and the test line; If bands appear on both the quality control line and the test line of the lateral flow chromatographic test strip, the to-be-detected sample contains or is a candidate to contain Liposcelis entomophila; If a band appears on the quality control line of the lateral flow chromatographic test strip and no band appears on the test line, the to-be-detected sample does not contain or is a candidate not to contain Liposcelis entomophila.

[0045] To solve the above technical problems, the present invention finally provides a method for differentiating or assisting in differentiating Liposcelis entomophila from other booklice.

[0046] The method for differentiating or assisting in differentiating Liposcelis entomophila from other booklice provided by the present invention includes the following steps: extracting the nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing multi-enzyme isothermal rapid amplification with the above-mentioned primer pair and probe; obtaining the multi-enzyme isothermal rapid amplification product; diluting the multi-enzyme isothermal rapid amplification product and dropping it into the sample adding hole of the lateral flow chromatographic test strip, and determining whether the booklice to be tested is Liposcelis entomophila or other booklice by observing whether bands appear on the quality control line and the detection line; if bands appear on both the quality control line and the detection line of the lateral flow chromatographic test strip, the booklice to be tested is or is a candidate for Liposcelis entomophila; if a band appears on the quality control line of the lateral flow chromatographic test strip and no band appears on the detection line, the booklice to be tested is or is a candidate for other booklice.

[0047] In any of the above methods, if no band appears on the quality control line of the lateral flow chromatographic test strip, the test result is invalid.

[0048] In any of the above methods, the reaction system for the multi-enzyme isothermal rapid amplification may be composed of 29.4 μL of A buffer, 2 μL of DNA template, 2 μL of primer - F (10 μM), 2 μL of primer - R (10 μM), 0.6 μL of probe (10 μM), 2.5 μL of B buffer, and 11.5 μL of sterile ultrapure water. The final concentrations of the primer - F and the primer - R in the reaction system are both 400 nM, and the final concentration of the probe in the reaction system is 120 nM.

[0049] In any of the above methods, the reaction conditions for the multi-enzyme isothermal rapid amplification may be constant temperature at 37 - 39 °C for 8 - 20 min, preferably constant temperature at 37 °C for 12 min.

[0050] In any of the above methods, the multi-enzyme isothermal rapid amplification product is diluted 10 - 20 times with sterile ultrapure water and then dropped into the sample adding hole of the lateral flow chromatographic test strip.

[0051] In any of the above methods, the nucleic acid may be genomic DNA.

[0052] Further, the genomic DNA can be obtained by extracting it using the booklice DNA crude extraction technique, or can also be obtained by using a blood / cell / tissue genomic DNA extraction kit.

[0053] Even further, the booklice DNA crude extraction technique may include the following steps: picking the booklice to be tested and placing it at the bottom of a centrifuge tube, adding sterile ultrapure water, and then grinding and crushing the insect body with a sterile pipette tip, toothpick, and grinding rod, centrifuging, heating in a 95 °C water bath or metal bath for 3 min, and centrifuging.

[0054] The preferred amount of the sterile ultrapure water added is 10 μL.

[0055] In any of the above-mentioned applications, kits or methods, the booklice to be detected may specifically be the eggs, nymphs or adults of the booklice to be detected.

[0056] In any of the above-mentioned applications, kits or methods, the sample to be detected includes various substances that can be damaged by the booklice Liposcelis bostrychophila, including various stored grains and other storage materials.

[0057] In any of the above-mentioned applications, kits or methods, the booklice Liposcelis bostrychophila include those from Beijing, China, those from Guyuan City, Ningxia Hui Autonomous Region, China, those from Changchun City, Jilin Province, China, and those from Tumote Left Banner and Sanggendale Town, Hohhot City, Inner Mongolia Autonomous Region, China.

[0058] In any of the above-mentioned applications, kits or methods, the other booklice may be species similar in morphology to the booklice Liposcelis bostrychophila, specifically at least one of the following booklice: Liposcelis mendax, Liposcelis bostrychophila, Liposcelis entomophila, Liposcelis brunnea, Liposcelis paeta, Liposcelis decolor, Liposcelis rufa.

[0059] Based on the multi-enzyme isothermal rapid amplification technology, the present invention designs two specific primers and a probe for identifying the booklice Liposcelis bostrychophila, and realizes the visual detection of the booklice Liposcelis bostrychophila through a lateral flow chromatography strip. The rapid identification method of the booklice Liposcelis bostrychophila established based on the multi-enzyme isothermal rapid amplification technology and the lateral flow chromatography strip technology can combine the crude DNA extraction technology of the booklice to realize the rapid identification of the eggs, nymphs and adults of the booklice Liposcelis bostrychophila. It not only gets rid of the dependence on instruments and equipment in the identification process, but also has the characteristics of being rapid, efficient, highly specific, sensitive, easy to operate and not affected by environmental conditions, and is suitable for rapid detection on site and is easy to popularize at the grass-roots level. The present invention provides a reliable technical basis for the prevention and control of the booklice Liposcelis bostrychophila, and is of great significance for protecting stored grains, preventing alien invasive organisms and assisting international trade, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a verification result diagram of the multi-enzyme isothermal rapid amplification of primer-F / R and the probe combined with the lateral flow chromatography strip. Among them, 1 is the 10th booklice Liposcelis bostrychophila; 2 is the 11th booklice Liposcelis bostrychophila; 3 is the 12th booklice Liposcelis bostrychophila; 4 is the 13th booklice Liposcelis bostrychophila; 5 is the 14th booklice Liposcelis bostrychophila; N is sterile ultrapure water.

[0061] Figure 2Figure showing the specific detection results of multi-enzyme isothermal rapid amplification combined with lateral flow chromatography strip using primers - F / R and probe. 1 is Liposcelis entomophila No. 10; 2 is Liposcelis entomophila No. 11; 3 is Liposcelis entomophila No. 12; 4 is Liposcelis entomophila No. 13; 5 is Liposcelis entomophila No. 14; 6 is Liposcelis decolor; 7 is Liposcelis bostrychophila (Group1); 8 is Liposcelis bostrychophila (Group2); 9 is Liposcelis bostrychophila (Group3); 10 is Liposcelis entomophila; 11 is Liposcelis brunnea; 12 is Liposcelis paeta; 13 is Liposcelis pallida; 14 is Liposcelis rufa; N is sterile ultrapure water.

[0062] Figure 3 Figure showing the sensitivity detection results of multi-enzyme isothermal rapid amplification combined with lateral flow chromatography strip using primers - F / R and probe. Among them, the DNA template concentrations of 1 - 7 are successively: 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng / μL, and N is sterile ultrapure water.

[0063] Figure 4 Figure showing the detection results of multi-enzyme isothermal rapid amplification combined with lateral flow chromatography strip technology using primers - F / R and probe for different life stages of Liposcelis entomophila. Among them, the DNA templates of 1 - 3 are respectively the crude extracted DNA of Liposcelis entomophila adults, nymphs, and eggs, and N is sterile ultrapure water. Specific Embodiments

[0064] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0065] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0066] In the following quantitative experiments, three repeated experiments are set, and the results are averaged.

[0067] The blood / cell / tissue genomic DNA extraction kit in the following examples is a product of Tiangen. A buffer, B buffer, and the reaction tube containing freeze-dried enzyme powder particles are all from the colloidal gold test strip type DNA isothermal rapid amplification kit, which is a product of Weifang Amp Future Biotechnology Co., Ltd., with the product number WLN8203KIT. The lateral flow chromatography test strip is a product of Weifang Amp Future Biotechnology Co., Ltd., named HybriDetect test strip, with the product number WLFS8201. The primers and probes were all synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0068] The numbers, species names, and collection location information of the tested booklice samples in the following examples are shown in Table 1. All tested booklice samples were identified by morphological and DNA barcode molecular identification methods before the test.

[0069] Table 1

[0070]

[0071] Example 1. Obtaining Primers, Probes, and Kits for Identifying Liposcelis entomophila

[0072] I. Design and Screening of Primers and Probes for Identifying Liposcelis entomophila

[0073] The primer for identifying Liposcelis entomophila designed and synthesized in the present invention is a primer pair composed of primer-F and primer-R, wherein the 5'-end of primer-R is labeled with biotin.

[0074] The probe for identifying Liposcelis entomophila designed and synthesized in the present invention is composed of a single-stranded DNA molecule shown in Sequence 3, T tetrahydrofuran, and a single-stranded DNA molecule shown in Sequence 4. Among them, the 5'-end of the probe is labeled with the fluorescent group FAM, and the 3'-end is modified with C3-Spacer.

[0075] The primer and probe sequences for identifying Liposcelis entomophila designed and synthesized in the present invention are shown in detail in Table 2.

[0076] Table 2

[0077]

[0078] II. Preparation of Kits for Identifying Liposcelis entomophila

[0079] The kit for identifying Liposcelis entomophila in the present invention includes the primers and probes designed in the above steps, the multi-enzyme isothermal rapid amplification reagent, and the lateral flow chromatography test strip.

[0080] III. Verification of Primers and Probes for Identifying Liposcelis entomophila

[0081] 1. The genomic DNA of the booklice samples was extracted using a Blood / Cell / Tissue Genomic DNA Extraction Kit to obtain the genomic DNA of the booklice samples. The booklice samples were Liposcelis entomophila numbered 10 - 14 in Table 1. The instars of the booklice samples were all adults.

[0082] 2. Using the genomic DNA of the booklice samples as a template, multi - enzyme isothermal rapid amplification was carried out in a reaction tube containing freeze - dried enzyme powder particles with the primers and probes designed in step 1 to obtain a multi - enzyme isothermal rapid amplification product.

[0083] The reaction system was 50 μL, consisting of 29.4 μL A buffer, 2 μL DNA template, 2 μL primer - F (10 μM), 2 μL primer - R (10 μM), 0.6 μL probe (10 μM), 2.5 μL B buffer, and 11.5 μL sterile ultrapure water. The final concentrations of primer - F and primer - R in the 50 μL reaction system were both 400 nM, and the final concentration of the probe in the 50 μL reaction system was 120 nM.

[0084] Reaction conditions: Incubate at 37 °C for 12 min.

[0085] According to the above method, the template was replaced with sterile ultrapure water, and other steps remained unchanged as a negative control.

[0086] 3. Take 5 μL of the amplification product, dilute it 20 - fold with sterile ultrapure water, and add 50 μL to the sample - adding well of the lateral flow chromatographic test strip, and observe the result within 5 min: If bands appear in both the test line and the quality control line of the lateral flow chromatographic test strip, the tested booklice is or is a candidate for Liposcelis entomophila; if no band appears in the test line of the lateral flow chromatographic test strip and a band appears in the quality control line, the tested booklice is not or is not a candidate for Liposcelis entomophila; if no band appears in the quality control line of the lateral flow chromatographic test strip, the test result is invalid.

[0087] The verification results of the multi - enzyme isothermal rapid amplification of primer - F / R and the probe combined with the lateral flow chromatographic test strip technology are shown in Figure 1 . Among them, 1 is Liposcelis entomophila No. 10, 2 is Liposcelis entomophila No. 11, 3 is Liposcelis entomophila No. 12, 4 is Liposcelis entomophila No. 13, 5 is Liposcelis entomophila No. 14, and N is sterile ultrapure water.

[0088] The results show that the method for identifying Liposcelis entomophila using the multi - enzyme isothermal rapid amplification technology combined with the lateral flow chromatographic test strip technology designed by the present invention with primer - F / R and the probe can successfully identify Liposcelis entomophila from five different geographical populations.

[0089] Example 2. Specificity experiment

[0090] The tested booklice 1 is Liposcelis entomophila No. 10.

[0091] The booklice to be tested 2 is Liposcelis entomophila numbered 11.

[0092] The booklice to be tested 3 is Liposcelis entomophila numbered 12.

[0093] The booklice to be tested 4 is Liposcelis entomophila numbered 13.

[0094] The booklice to be tested 5 is Liposcelis entomophila numbered 14.

[0095] The booklice to be tested 6 is Liposcelis decolor numbered 1.

[0096] The booklice to be tested 7 is Liposcelis bostrychophila numbered 2.

[0097] The booklice to be tested 8 is Liposcelis bostrychophila numbered 3.

[0098] The booklice to be tested 9 is Liposcelis bostrychophila numbered 4.

[0099] The booklice to be tested 10 is Liposcelis entomophila numbered 5.

[0100] The booklice to be tested 11 is Liposcelis brunnea numbered 6.

[0101] The booklice to be tested 12 is Liposcelis paeta numbered 7.

[0102] The booklice to be tested 13 is Liposcelis decolor numbered 8.

[0103] The booklice to be tested 14 is Liposcelis rufa numbered 9.

[0104] The developmental stages of the booklice samples to be tested are all adults.

[0105] The following steps are carried out for each booklice to be tested:

[0106] 1. Extract the genomic DNA of the booklice to be tested using a Blood / Cell / Tissue Genomic DNA Extraction Kit.

[0107] 2. Using the genomic DNA of the booklice to be tested extracted in step 1 as a template, perform multi-enzyme isothermal rapid amplification in a reaction tube containing enzyme powder freeze-dried particles with primers -F / R and the corresponding probe to obtain a multi-enzyme isothermal rapid amplification product.

[0108] The reaction system is 50 μL, consisting of 29.4 μL A buffer, 2 μL DNA template, 2 μL primer -F (10 μM), 2 μL primer -R (10 μM), 0.6 μL probe (10 μM), 2.5 μL B buffer, and 11.5 μL sterile ultrapure water.

[0109] Reaction conditions: Constant temperature at 37 °C for 12 min.

[0110] According to the above method, replace the template with sterile ultrapure water, and keep other steps unchanged as the negative control.

[0111] According to the above method, replace the template with the genomic DNA of Liposcelis bostrychophila (numbered 10, 11, 12, 13, 14), and keep other steps unchanged as the positive control.

[0112] 3. Take 5 μL of the amplification product, dilute it 20-fold, and add 50 μL to the sample application hole of the lateral flow chromatographic test strip, and observe the result within 5 minutes.

[0113] The specific detection results of primer - F / R and the probe are shown in Figure 2 . Among them, 1 - 14 are Liposcelis bostrychophila to be detected 1 - 14; N is sterile ultrapure water.

[0114] The results show that only the lateral flow chromatographic test strip with the diluted solution of the amplification product of Liposcelis bostrychophila shows bands at both the quality control line and the detection line, while for other Liposcelis bostrychophila and sterile ultrapure water, only bands at the quality control line can be observed and no bands at the detection line can be observed. Thus, it can be seen that the method for identifying Liposcelis bostrychophila using the primer - F / R and the probe in combination with the multi - enzyme isothermal rapid amplification technology and the lateral flow chromatographic test strip technology designed by the present invention has good specificity.

[0115] Example 3: Sensitivity experiment

[0116] 1. Extract the genomic DNA of Liposcelis bostrychophila (adult) numbered 13 in Liposcelis bostrychophila using the Blood / Cell / Tissue Genomic DNA Extraction Kit.

[0117] 2. Gradient - dilute the genomic DNA of Liposcelis bostrychophila extracted in step 1 with sterile water to obtain diluted solutions with the genomic DNA concentrations of Liposcelis bostrychophila being 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng / μL.

[0118] 3. Using the diluted solutions obtained in step 2 as templates, perform multi - enzyme isothermal rapid amplification in reaction tubes containing enzyme powder freeze - dried particles with primer - F / R and the probe to obtain multi - enzyme isothermal rapid amplification products.

[0119] The reaction system is 50 μL, which consists of 29.4 μL of A buffer, 2 μL of DNA template, 2 μL of primer - F (10 μM), 2 μL of primer - R (10 μM), 0.6 μL of probe (10 μM), 2.5 μL of B buffer, and 11.5 μL of sterile ultrapure water.

[0120] Reaction conditions: Constant temperature at 37°C for 12 min.

[0121] According to the above method, replace the template with sterile ultrapure water, and keep other steps unchanged as the negative control.

[0122] 4. Take 5 μL of the amplification product, dilute it 20-fold, and pipette 50 μL into the sample application well of the lateral flow chromatographic test strip, and observe the result within 5 min.

[0123] If bands appear on both the quality control line and the test line of the lateral flow chromatographic test strip, it indicates that the multi-enzyme isothermal rapid amplification product is positive, indicating that the corresponding DNA template content in the reaction system can be detected; if a band appears on the quality control line of the lateral flow chromatographic test strip but no band appears on the test line, it indicates that the corresponding DNA template content in the reaction system cannot be detected; if no band appears on the quality control line of the lateral flow chromatographic test strip, the experimental result is invalid.

[0124] The sensitivity detection results of primer - F / R and the probe are shown in Figure 3 . Among them, the DNA template concentrations of 1 - 7 are successively: 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng / μL, and N is sterile ultrapure water.

[0125] The results show that: the sensitivity of primer - F / R and the probe for identifying Liposcelis entomophila is 1.0 ng / μL. Thus, it can be seen that the method for identifying Liposcelis entomophila by using primer - F / R and the probe in combination with the multi-enzyme isothermal rapid amplification technology and the lateral flow chromatographic test strip technology designed by the present invention has high sensitivity.

[0126] Example 4. Identification of different life stages of Liposcelis entomophila

[0127] 1. Extract the genomic DNA of single eggs, single nymphs, and single adults of Liposcelis entomophila numbered 11 by using the Liposcelis DNA crude extraction technology. The specific steps are as follows: Pick a single adult Liposcelis entomophila, a single nymph Liposcelis entomophila, and a single egg and place them at the bottom of a 1.5 mL centrifuge tube, add 10 μL of sterile ultrapure water, grind the insect body into powder with a sterile pipette tip, toothpick, and grinding rod, centrifuge, heat in a 95°C water bath or metal bath for 3 min, and then centrifuge.

[0128] 2. Take the genomic DNA of Liposcelis entomophila in different life stages (eggs, nymphs, adults) as templates respectively, and perform multi-enzyme isothermal rapid amplification in a reaction tube containing freeze-dried enzyme powder particles by using primer - F / R and the probe to obtain the multi-enzyme isothermal rapid amplification product.

[0129] The reaction system is 50 μL and consists of 29.4 μL of A buffer, 2 μL of DNA template, 2 μL of primer-F (10 μM), 2 μL of primer-R (10 μM), 0.6 μL of probe (10 μM), 2.5 μL of B buffer, and 11.5 μL of sterile ultrapure water.

[0130] Reaction conditions: Constant temperature at 37 °C for 12 min.

[0131] According to the above method, replace the template with sterile ultrapure water, and keep other steps unchanged as the negative control.

[0132] 3. Take 10 μL of the amplification product, dilute it 10-fold, and add 50 μL dropwise to the sample addition hole of the lateral flow chromatographic test strip, and observe the result within 5 min.

[0133] The detection results of different life stages of Liposcelis bostrychophila using primer-F / R and probe are shown in Figure 4 . Among them, the DNA templates of 1 - 3 are the crude extracted DNAs of Liposcelis bostrychophila adults, nymphs, and eggs respectively, and N is sterile ultrapure water.

[0134] The results show that when the samples are the eggs, nymphs, and adults of Liposcelis bostrychophila, both the quality control line and the detection line of the lateral flow chromatographic test strip with the diluted multi-enzyme isothermal rapid amplification product show bands; when the sample is the negative control, the quality control line of the lateral flow chromatographic test strip shows a band while the detection line does not show a band. Thus, it can be seen that the method for identifying Liposcelis bostrychophila using primer-F / R and probe in combination with DNA crude extraction technology, multi-enzyme isothermal rapid amplification technology, and lateral flow chromatographic test strip technology designed by the present invention can be used for the rapid identification of Liposcelis bostrychophila in different life stages.

[0135] The above has elaborated on the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including those that deviate from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A kit of reagents for identifying or assisting in the identification of Liposcelis bostrychophila, said kit of reagents comprising a primer pair for identifying or assisting in the identification of Liposcelis bostrychophila; the primer pair for identifying or assisting in the identification of Liposcelis bostrychophila consists of primer-F and primer-R; The primer-F is as follows a1) or a2): a1) A single-stranded DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing; a2) A single-stranded DNA molecule which is obtained by substituting and / or deleting and / or adding one or several nucleotides to SEQ ID NO: 1 and has the same function as SEQ ID NO: 1; The primer-R is as follows a3) or a4): a3) A single-stranded DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing; a4) A single-stranded DNA molecule which is obtained by substituting and / or deleting and / or adding one or several nucleotides to SEQ ID NO: 2 and has the same function as SEQ ID NO:

2.

2. The kit of reagents according to claim 1, wherein: The kit of reagents further comprises a probe for identifying or assisting in the identification of Liposcelis bostrychophila; the probe consists of a single-stranded DNA molecule shown in SEQ ID NO: 3, T tetrahydrofuran and a single-stranded DNA molecule shown in SEQ ID NO:

4.

3. The kit of reagents according to claim 1 or 2, characterized in that: The molar ratio of the primer-F, the primer-R and the probe is 10:10:

3.

4. Use of the kit of reagents according to any one of claims 1-3 in any one of the following b1)-b6): b1) Identifying or assisting in the identification of whether a to-be-detected booklice is Liposcelis bostrychophila; b2) Preparing a product for identifying or assisting in the identification of whether a to-be-detected booklice is Liposcelis bostrychophila; b3) Detecting or assisting in the detection of whether a to-be-detected sample contains Liposcelis bostrychophila; b4) Preparing a product for detecting or assisting in the detection of whether a to-be-detected sample contains Liposcelis bostrychophila; b5) Distinguishing or assisting in the distinction between Liposcelis bostrychophila and other booklice; b6) Preparing a product for distinguishing or assisting in the distinction between Liposcelis bostrychophila and other booklice.

5. A kit containing the kit of reagents according to any one of claims 1-3; the function of the kit is any one of the following c1)-c3): c1) Identifying or assisting in the identification of whether a to-be-detected booklice is Liposcelis bostrychophila; c2) Detecting or assisting in the detection of whether a to-be-detected sample contains Liposcelis bostrychophila; c3) Distinguishing or assisting in the distinction between Liposcelis bostrychophila and other booklice.

6. The kit according to claim 5, wherein: The kit further comprises a multi-enzyme isothermal rapid amplification reagent and a lateral flow chromatographic test strip.

7. A preparation method of the kit according to claim 5 or 6, which is as follows d1) or d2): d1) Separately packaging the primer pair and the probe in the kit of reagents according to claims 1-3; d2) Proportionally pairing and mixing the primer pair and the probe in the kit of reagents according to claims 1-3.

8. A method for identifying or assisting in the identification of whether a to-be-detected booklice is Liposcelis bostrychophila, comprising the following steps: extracting the nucleic acid of the to-be-detected booklice, using the nucleic acid of the to-be-detected booklice as a template, performing multi-enzyme isothermal rapid amplification by using the primer pair and the probe described in claims 1-3 to obtain a multi-enzyme isothermal rapid amplification product; diluting the multi-enzyme isothermal rapid amplification product and dropping it into the sample adding hole of the lateral flow chromatographic test strip, and judging whether the to-be-detected booklice is Liposcelis bostrychophila by observing whether bands appear on the quality control line and the detection line; if bands appear on both the quality control line and the detection line of the lateral flow chromatographic test strip, the to-be-detected booklice is or is a candidate for Liposcelis bostrychophila; if a band appears on the quality control line of the lateral flow chromatographic test strip and no band appears on the detection line, the to-be-detected booklice is not or is not a candidate for Liposcelis bostrychophila.

9. A method for detecting or assisting in detecting whether a sample to be tested contains Liposcelis bostrychophila, comprising the following steps: extracting the nucleic acid of the sample to be tested, using the nucleic acid of the sample to be tested as a template, and performing multi-enzyme isothermal rapid amplification with the primer pairs and probes described in claims 1-3 to obtain a multi-enzyme isothermal rapid amplification product; diluting the multi-enzyme isothermal rapid amplification product and dropping it into the sample application hole of a lateral flow chromatographic test strip, and determining whether the sample to be tested contains Liposcelis bostrychophila by observing whether bands appear on the quality control line and the detection line; If bands appear on both the quality control line and the test line of the lateral flow chromatographic test strip, the sample to be tested contains or is candidate to contain Liposcelis bostrychophila Badonnel; If a band appears on the quality control line of the lateral flow chromatographic test strip and no band appears on the test line, the sample to be tested does not contain or is candidate not to contain Liposcelis bostrychophila Badonnel.

10. A method for differentiating or assisting in differentiating Liposcelis entomophila from other booklice, comprising the following steps: extracting the nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing multi-enzyme isothermal rapid amplification with the primer pairs and probes described in claims 1-3; obtaining a multi-enzyme isothermal rapid amplification product; diluting the multi-enzyme isothermal rapid amplification product and dropping it into the sample addition hole of a lateral flow chromatographic test strip, and determining whether the booklice to be tested is Liposcelis entomophila or other booklice by observing whether bands appear on the quality control line and the detection line; If bands appear on both the quality control line and the test line of the lateral flow chromatographic test strip, the lice to be tested are or are candidate to be Liposcelis bostrychophila Badonnel; If a band appears on the quality control line of the lateral flow chromatographic test strip and no band appears on the test line, the lice to be tested are or are candidate to be other Liposcelis.

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