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Delivery: <= 4 days. True-PDF full-copy in English will be manually translated and delivered via email. SN/T 1105-2019: Detection and identification of Coptotermes curvignathus Holmgren Status: Valid SN/T 1105: Historical versions
Basic dataStandard ID: SN/T 1105-2019 (SN/T1105-2019)Description (Translated English): Detection and identification of Coptotermes curvignathus Holmgren Sector / Industry: Commodity Inspection Standard (Recommended) Classification of Chinese Standard: B16 Word Count Estimation: 17,197 Date of Issue: 2019-09-03 Date of Implementation: 2020-03-01 Older Standard (superseded by this standard): SN/T 1105-2014 Regulation (derived from): Natural Resources Department Announcement No. 7 of 2019 Issuing agency(ies): General Administration of Customs SN/T 1105-2019: Detection and identification of Coptotermes curvignathus Holmgren---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.(Everyone's quarantine identification method) ICS 65.002.01B16 People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard SN/T 11105-2019 instead of Sn/T 1105-2014 Everyone termite quarantine identification method Published.2019-2009-03 2020-03-01 implementation Published by the General Administration of Customs of the People's Republic of China ????? ????, ????, ?? ForewordThis standard was drafted in accordance with the rules given in GB/T 1.1-2009. This standard replaces Sn/T 1105-2014 "Everybody's termite quarantine and identification method". Compared with SN/T 1105-2014, the main technical changes of this standard are as follows. --- Removed normative references; --- Modified terms and definitions; --- Modified equipment and reagents, integrated morphology and molecular biology; --- Increase the content of on-site quarantine to facilitate the detection of termites; --- Increased identification feature measurement locations; --- Added the host and distribution of termites in Appendix A; --- Added Appendix B to the research overview of the world termite, and clarified the synonym of the termite genus. Delete the original standard approximation Synonyms of termites, Philippine island termites are synonymous with Gerst's stage termites, and Sinabon's termites are synonymous with termites Synonym --- Modified the search form for appendix C termites and their approximate species; --- Added Appendix E color pictures of termites; --- Added Appendix F color pictures of similar termites. This standard is proposed and managed by the General Administration of Customs of the People's Republic of China. This standard was drafted. Xiamen Customs of the People's Republic of China, Gongbei Customs of the People's Republic of China. The main drafters of this standard. Pang Jin, Huang Pengying, Fang Zhipeng, Fang Yuanwei, Lin Zhenji, Yao Xiangrong, Dong Wenguang, Zhang Weidong, Liao Li. The release of the version replaced by this standard is. --- SN/T 1105-2014. SN/T 1105-201919 ????? ????, ????, ?? Everyone termite quarantine identification method1 ScopeThis standard applies to the quarantine and identification of termites.2 Terms and definitionsThe following terms and definitions apply to this document. 2.1 The termite is recessed or protruding at the confluence of the seam of the head cover, which is the opening of the forehead. 2.2 Round opening in the middle of the forehead. 2.3 2.4 2.5 2.6 2.7 Soldier and worker ants refer to the widest part of the head, and winged adults refer to the width of the head including compound eyes. 2.8 2.9 2.10 The maximum width of the pronotum. SN/T 1105-201919 ????? ????, ????, ??3 Basic information on termitesSpreading of bedding materials such as sawn wood and wood packaging spread. The host, distribution and biology are shown in Appendix A. Overview of World Termite Research See Appendix B. Because there are not many similar species of milk termites in the Americas, Africa, and Oceania, they are generally easier to identify. There are many types of milk termites in Asia, which are difficult distinguish. See Appendix C for the retrieval tables of termites and other important milk termites in Asia.4 Method stepsAccording to the damage of termites, you can obtain eggs, soldier ants, worker ants, or adults at the quarantine site or where suspected termites are. Present If field quarantine finds soldier termites with termites, they are identified based on morphological characteristics. If termite soldier ants are not found, but have eggs, worker ants or winged adults When you wait, the conventional PCR detection method and real-time fluorescent PCR detection method of the termite-specific amplification primers of everyone can be accurate and fast. Quickly distinguish everyone from termites, Gerst milk termites, Taiwan milk termites, and South Asia milk termites.5 Equipment and reagents5.1 Equipment Microscope, dissecting mirror, bark shovel, handsaw, chainsaw, iron cone, screwdriver, small axe, scalpel, dissecting needle, forceps, petri dish, writing brush, Worm tube, finger tube, micrometer; PCR amplification instrument, real-time fluorescence PCR instrument, electrophoresis instrument, gel imaging system, centrifuge, desktop centrifuge, nuclear Acid protein analyzer, oven, autoclave, electronic balance, mortar, shaker, water bath, vortex shaker, refrigerator, micro-sampler (0.5 μL, 2 μL, 10 μL, 20 μL, 100 μL,.200 μL, 1000 μL), PCR reaction tube (0.2 mL, 0.5 mL). 5.2 Reagents 75% ethanol, double distilled water, liquid nitrogen, proteinase K (20mg/mL), RNA digestion enzyme, Tris saturated phenol, PremexExTa (2 ×), chloroform, isoamyl alcohol, isopropanol, 75% ethanol, 95% ethanol, 10 × PCR buffer, deoxyribonucleoside triphosphates (dNTPs), Tazyme, DL2000 Marker, agarose, ethidium bromide (EB), primer probe, insect genome DNA extraction kit. Extract A. 1% SDS, 50 mmol/L Tris · Cl (PH8.0), 25 mmol/L NaCl, 25 mmol/LEDA (PH 8. 0). 6 On-site inspection 6.1 Check for holes Tap the ends and middle of the log or wood with an iron cone to hear if there is a hollow echo and find out if there are holes. If it makes an unusual sound, The hole should be pried open and carefully checked for termites. 6.2 Checking the exposed traces of termites Check whether the quarantine materials such as logs and wood cubes have termite sludge (ie, termite excrement and soil accumulation of ants), fly holes (this hole is winged into SN/T 1105-201919 ????? ????, ????, ?? Insects breeding fly holes, which are often sealed by soil) and vent holes (ie, pinpoint-shaped holes with a diameter of about 1mm outside the ant nest, usually more than ten There are dozens of holes, sometimes with fine soil and guards by soldiers and ants). Use a saw to cut the wood to check the signs of danger, if necessary, Sampling this paragraph back to indoor quarantine. When the log has bark, you need to lift the bark with a shovel or screwdriver and observe whether the inside of the bark and the xylem of the log There are termite hazards or bugs. 6.3 Finding the Nest On the scene, carefully search the nests of suspicious woods with exposed termite activity. If termite activity is found, collect specimens in time to Prepare for laboratory identification.7 Laboratory identification7.1 Specimen processing The specimen was placed in a petri dish and washed with 75% ethanol, and the wood chips and dirt attached to the insect body were gently removed with a fine brush. Stereomicroscopy Use a dissecting needle to straighten the worm body under the microscope, so that the main characteristics of the identification are on a plane, especially the soldiers need to separate the upper jaw after the worm body softens. 7.2 Morphological characteristics measurement Specimen bodies were observed with a stereo microscope with a micrometer, and important identification characteristics were measured. 10 (except for a small number of specimens). Soldier ant specimen measurements include. head to palate, head widest, head height, left palate length, long hole diameter, short hole Diameter, widest posterior condyle, narrowest posterior condyle, width of pronotum, prostrate length, and number of antennae. Winged adult specimen measurements include body length (not With wings), head to upper lip, head to upper jaw, head wide with eyes, compound eyes long diameter, compound eyes short diameter, compound eyes to head lower edge, monocular long diameter, monocular short Diameter, fore-wing length (without scales), pronotum length, pronotum width, hindfoot tibial length, and number of antennae nodes. Units are millimeters (mm). 7.3 Records and calculations Record all measured features, and convert the measured values according to the multiples of the stereo microscope, uniformly converted to millimeters (Mm). At the same time, the average of all measured individuals is calculated. 7.4 Morphological identification 7.4.1 Identification of Milky termites 7.4.1.1 Morphological characteristics of soldier ants The head is ovate, and the anterior segment is obviously narrowed. The ridge is located at the front of the head and extends as a short tube. The tooth shape is notched, the inner edge is smooth and toothless, the antennae are 13 to 17 knots, and the tip of the upper lip swallows. The pronotum is flat and narrower than the head. 7.4.1.2 Morphological characteristics of winged adults The head is broadly ovate, the base of the rear lip is extremely short and flat, and the antennae are 18 to 25 knots. The pronotum is flat, narrower than the head, and the front wings The scales are larger than the hind wing scales and cover the hind wing scales. The veins of the wings have a very shallow reticulate pattern, and the wings have mask hairs. The veins emerge from the base of the tibial vein, and the midrib is very close to the elbow. 7.4.2 Identification of termites 7.4.2.1 Morphological characteristics of worker ants The body length is from 4.10mm to 5.05mm. The head is nearly round, light brown, with a head width of 1.30mm to 1.75mm, and a head length of 0.85mm to 1.05mm, antennae 14 to 15 knots. The width of the pronotum is 0.60mm ～ 0.85mm, the leading edge is slightly raised, there is a nick in the center. SN/T 1105-201919 ????? ????, ????, ?? Belly is milky white, sparsely yellowish short-haired. 7.4.2.2 Morphological characteristics of soldier ants The head shell is dark yellow, the upper jaw is dark brown, and the chest, abdomen and feet are pale yellow. The head shell is sparsely hairy, with a pair of end hairs on the upper lip end, one on each side of the palate 1 bristle, there is also a thick bristle between the palate and the antennae, about 20 short hairs in the middle of the pronotum. Oval head shell, widest behind the head Half, long and thin palate, saber-shaped, strong end of the jaw, a deep recess at the base of the left upper jaw, with a small dent in the front, tongue-shaped upper lip, slightly longer than Wide, slightly pointed at the ends, translucent at the ends of the lips, large, round calyx holes, oblique openings on the side, and the upper part slightly backward. Antennae 15 knots to 16 knots, generally 16 Sections 3, 4 are short and almost equal, and section 2 is slightly longer than or equal to section 3. Prosthetic dorsum plate ingot-shaped, wider than long, with central anteroposterior edge Slightly concave, rounded on the front side, and obliquely trailing on both sides (see Appendix D, Appendix E, and Appendix F). 7.4.2.3 Morphological characteristics of winged adults The total length is 16mm ~ 17mm, the wing length is 13mm ~ 14mm, the body length (excluding wing) is 7.5mm, the head is nearly round, dark brown, and the head is long 1.15mm. The tentacles are yellow-brown, 21 knots, and the second, third, and fourth knots are shorter than the other knots, and the third knot is the shortest. Compound eyes large, near-circular, monocular oval shape. The distance between the antennae and the compound eye is smaller than the distance between the antennae and the compound eye, and the distance between the single eye and the compound eye is less than the width of the single eye. The front edge of the pronotum Concave, the center of the trailing edge is recessed forward. Pronotum brown, densely yellow-brown long-haired. Fore-wing scales are larger than hind-wing scales. It is colored hairy, with the forelimb midline protruding independently from the shoulder seam, and the elbow vein having 4 to 10 branches. Hind wing midribs are from the base of the radial veins, and the elbow veins have 8 There are 10 to 10 branches, and the wing veins vary greatly among different individuals. 7.5 Molecular biology identification 7.5.1 Isolation of Termite Genomic DNA Take one termite (worker ants, soldier ants, winged adults, etc.) and rinse it in double-distilled water, blot it with absorbent paper, and remove the attachments on its surface under a dissecting microscope. And abdominal contents to prevent protozoa contamination. Place it in a 1.5 ml Eppendorf tube, add 40 μL of extraction solution A, put it in a -70 ° C refrigerator, remove it after 4 min, mash it with a toothpick, homogenize, add an equal volume of 3 mmol/L potassium acetate (PH7.2), and ice Place on top for 1h, add Add an equal volume of phenol/chloroform, mix, centrifuge at 2,000 g for 10 min, take the supernatant, add 2 times the volume of frozen absolute ethanol, mix and place in the refrigerator for 1 h Precipitate DNA; centrifuge at 1,500 g for 15 min, discard the supernatant, add 1 mL of 75% ethanol to wash the pellet, centrifuge at 2,000 g for 1 min, and dry the ethanol Then, the precipitate was dissolved with 100 μL double-distilled water, and the concentration and purity of DNA were measured by a microspectrophotometer. Store at -20 ° C until use. Genomic DNA can also be extracted by kit method. 7.5.2 DNA quality check The purity and concentration of DNA were measured with a nucleic acid protein analyzer, and the absorption values at 260 nm and 280 nm were obtained, respectively. Purity and concentration, the calculation formula is as follows. DNA purity = OD260/OD280 DNA concentration = 50 × OD260 μg/mL The OD260/OD280 ratio of the PCR grade DNA solution should be 1.7 to 1.9. 7.5.3 Routine PCR detection 7.5.3.1 Primers The sequence of the PCR primers for termites is shown in Table 1. SN/T 1105-201919 ????? ????, ????, ?? Table 1 Primer sequences Type primer name primer sequence amplified fragment Forward primer Reverse primer CC-1F CC-1R 5'-AGCGACCGCGCTCTCGCGTGCG-3 ' 5'-CCCATCTCTCGCGCGCCCGCGCGCT-3 '254bp 7.5.3.2 Reaction system See Table 2 for the conventional PCR reaction system. Table 2 Reaction system Reagent volume/μL 10 × PCR buffer (with Mg2 +) 2.5 μL dNTP mixture (10 mmol/L) 2 μL Forward primer and reverse primer (10 μmol/L) each 1 μL Tazyme (5U/μl) 0.2μL Template DNA 5μL ddH2O supplemented to a total volume of 25 μL 7.5.3.3 Reaction conditions Pre-denatured at 94 ° C for 5 min; denatured at 94 ° C for 1 min, annealing extended at 72 ° C for 1 min, 30 cycles; finally, extended at 72 ° C for 5 min. 7.5.3.4 Setting of negative control, positive control and blank control The DNA templates of other species of Pterotermes were used as negative controls, and the DNA of every termite was used as a positive control, and sterilized deionized water was used. For blank control, make 2 replicates each. 7.5.3.5 Agar gel electrophoresis detection The size of the amplified target fragments was checked by agarose gel electrophoresis on conventional PCR products. Take 5μL of PCR product, add 1μL of 10 × PCR buffer, and use DL2000 Marker as molecular weight marker, electrophoresis on a 1.5% agarose gel containing DNA staining agent for 30min (90V), check whether the amplification is on the gel image analyzer A target segment of the expected size is taken, and the results are recorded and recorded. 7.5.4 Real-time fluorescent PCR detection 7.5.4.1 Primers and probes The real-time fluorescent PCR primers and probe sequences of the termites are shown in Table 3. Table 3 Primer and probe sequences Type primer name primer sequence amplified fragment Forward primer Reverse primer Probe CC-2F CC-2R CC-TT 5'-AGCGCCCGCGCGCGCCCGAGAGTGTG-3 ' 5'-CCGAGACTCTCTCTGTGTTTTC-3 ' 5'-FAM-ACGACACGCGCTCTGTCTATCTCTCG-TAMRA-3 ' 155bp SN/T 1105-201919 ????? ????, ????, ?? 7.5.4.2 Reaction system The real-time fluorescence reaction system of termites is shown in Table 4. Table 4 Reaction system Reagent volume/μL PremExTaz (2 ×) 12.5 μL Forward primer and reverse primer (10 μmol/L) each 0.5 μL Probe (10 μmol/L) 0.25 μL Template DNA 2μL ddH2O supplemented to a total volume of 25 μL 7.5.4.3 Reaction conditions Pre-denatured at 94 ° C for 1 min; denatured at 94 ° C for 15 s, and annealed at 60 ° C for 1 min, 40 cycles. 7.5.4.4 Setting of negative control, positive control and blank control Same as 7.5.3.4.8 result judgment8.1 Morphological methods Based on the morphological characteristics of soldier ants, the identification characteristics of winged adults can be used as a reference, and they can be judged as termites if they conform to 7.4.2.2. 8.2 Molecular biology methods 8.2.1 Routine PCR detection Electrophoresis imaging results, if the positive control sample and the test sample both show a specific DNA product band of about 254 bp, and the negative There is no DNA product band in the control sample. The above experiment was repeated two or more times. If the results are consistent, it can be determined that the type of sample is everyone. termite. 8.2.2 Real-time fluorescent PCR detection When the results of the positive control, negative control, and blank control are normal, then. --- The detection Ct value is less than or equal to 36, the test result is positive and the sample type is everyone termites; --- The detection Ct value is greater than or equal to 40, and the test result is negative to determine that the sample type is not a termite; --- The detection Ct value is between 36 and 40, it is necessary to redo the real-time fluorescence PCR.9 Specimen preservationSpecimens collected for morphological identification should be stored in wide-mouth bottles containing 75% ethanol, and the source of the goods should be indicated. Master, collection time, place and collector, storage period is 6 months to 1 year; specimens used for molecular biological identification are soaked with 100% ethanol, Store in -70 ℃ refrigerator for future use. SN/T 1105-201919 ????? ????, ????, ??Appendix A(Informative appendix) Everyone termite host, distribution and damage A. 1 host Albizia, Soybean, Cashew, Araucaria, Jackfruit, Kapok, Olive, Jibe, Eucalyptus, Hevea, Twin Leaf Bean, Mango, Wood butterfly, Pinus, Caribbean pine, Sumatra pine, Salix, Red eucalyptus, Salo, benzoin, mahogany, teak, etc. A. 2 Distribution Asia. Singapore, Malaysia, Thailand, Myanmar, Vietnam, Indonesia, Cambodia, Brunei, India, Philippines, Papua New Guinea Asia, Papua New Guinea, South Korea. Europe. Germany. Africa. South Africa A. 3 harm Everyone termites are social polymorphic insects. The same nest group has different grades of differentiation, including ants, queens, worker ants, soldier ants, and reproduction. They are divided into ants, and each has their own responsibilities. Among them, soldier ants have strange changes and have obvious discriminative value in taxonomy. The worm is a terrestrial termite that can Nesting in wood, you can also nest in the soil. The hidden lifestyle, the occurrence of various grades and the development of the entire nest group are in the nest. Nesters in the wood are mostly in the hollow of the wood. Small holes, vent holes or openings for worker ants are sometimes visible on the surface of the damaged wood, and are common There are excreta or dirt. Termites invaded from the roots of the ground, eating stems. The ant can cause severe damage to a variety of economic forest trees, can damage live trees, and erode Ground cables, instruments and equipment in the forest, damage to the house and various types of wood packaging. SN/T 1105-201919 ????? ????, ????, ??Appendix B(Informative appendix) Overview of World Termite Research According to the "World Term Essay Collection" written......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of SN/T 1105-2019_English be delivered?Answer: Upon your order, we will start to translate SN/T 1105-2019_English as soon as possible, and keep you informed of the progress. The lead time is typically 2 ~ 4 working days. The lengthier the document the longer the lead time.Question 2: Can I share the purchased PDF of SN/T 1105-2019_English with my colleagues?Answer: Yes. The purchased PDF of SN/T 1105-2019_English will be deemed to be sold to your employer/organization who actually pays for it, including your colleagues and your employer's intranet.Question 3: Does the price include tax/VAT?Answer: Yes. 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