LU101603B1 - Primers, method and kit for assisting identification of Pomacea Canaliculata - Google Patents
Primers, method and kit for assisting identification of Pomacea Canaliculata Download PDFInfo
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- LU101603B1 LU101603B1 LU101603A LU101603A LU101603B1 LU 101603 B1 LU101603 B1 LU 101603B1 LU 101603 A LU101603 A LU 101603A LU 101603 A LU101603 A LU 101603A LU 101603 B1 LU101603 B1 LU 101603B1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses primers, a method and a kit for assisting identification of Pomacea canaliculata. The primers refer to a primer pair PcanF and PcanR for amplifying a mitochondrial cytochrome oxidase subunit I gene of an apple snail. With the application of the specific primer pair of the invention, the Pomacea canaliculata can be identified at a molecular level without being affected by the development status and integrity of an individual sample, thereby achieving the advantages of high efficiency, convenience, accuracy, and high sensitivity. The invention solves the problem encountered in the species identification of the apple snail, which has puzzled plant protection and port quarantine departments for a long time. The invention may have a broad application in the plant protection departments and quarantine departments.
Description
[0001] 1. Technical Field
[0002] The invention belongs to the field of biotechnology, and particularly relates to primers, a method and a kit for assisting identification of Pomacea canaliculata(Pomacea canaliculata, P. canaliculata).
[0003] 2. Description of Related Art
[0004] Apple snails, belonging to the genus Pomacea (Perry, 1810), the family Ampullariidae, the order Caeogastropoda, the class Gastropoda, are world-recognized invasive aquatic organisms. Pomacea canaliculata, native to South America, has invaded many countries and regions Asia and North America. Since the P. canaliculata was introduced and colonized in China in the early 1980s, it has become one of the most dangerous exotic species firstly announced in China. At present, the P canaliculata has spread to 14 provinces in southern China and has caused serious damages. As omnivores, the apple snails may damage a variety of aquatic crops, causing losses to the rice production in southern China. Also, they occupy the ecological niches and break the balance of the aquatic ecosystem. In addition, the apple snails are intermediate hosts of a variety of parasites, and their spreading directly affect the prevalent of parasitic diseases.
[0005] Different types of pests have different physiological, ecological, and hazard characteristics, leading to differences in the degrees of harm, environmental adaptability, spreading speed and other aspects. Therefore, accurate species identification provides a basis for conducting biological researches and selectingappropriate control measures. The group of golden apple snails includes many LU101603 morphologically similar species, and is one of the most difficult species for morphological identification in the family Ampullariidae. Traditionally, the apple snails are mainly identified through morphological characteristics of adult apple snails and their eggs. However, the intraspecific morphological variations of apple snails are very susceptible due to factors such as food sources, external environment, and developmental phases, resulting in great difficulty and uncertainty in morphological identification. The development of the molecular biology technology in recent years has made it possible to test the species of the apple snails rapidly and accurately. Molecular identification is accurate, rapid and simple and is easy to standardize, which greatly makes up for the defects of the traditional morphological identification.
[0006] An object of the invention is to provide a specific primer pair and a kit for assisting identification of P. canaliculata. The kit includes the above-mentioned specific primer pair, a PCR reaction system, an amplification program, and a product testing | method.
[0007] In view of the practical needs of plant protection, customs and market supervision departments for species identification of the applies snails, the invention | designs and screens a specific primer pair for identifying the P. canaliculata and devises a method for identifying and testing the P. canaliculata, for identifying the P.canaliculata rapidly, conveniently and accurately.
[0008] The first object of the invention is to provide primers for assisting identification of P canaliculata, wherein the primers refer to a primer pair PcanF and PcanR for amplifying a mitochondrial cytochrome oxidase subunit I gene of a applesnail, with sequences as follows: LU101603
[0009] PcanF (SEQ ID NO.1): 5”-GAGATGACCAGCTTTATAATGTC-3°;
[0010] PcanR (SEQ ID NO.2): 5’-AAGAACCGGCAATGATAAGAGC-3’.
[0011] The second object of the invention is to provide a kit for assisting identification of P. canaliculata, wherein the kit includes a primer pair PcanF and PcanR for amplifying a mitochondrial cytochrome oxidase subunit I gene of a golden apple snail, with sequences as follows:
[0012] PcanF (SEQ ID NO.1): 5’-GAGATGACCAGCTTTATAATGTC-3’;
[0013] PcanR (SEQ ID NO.2): 5’-AAGAACCGGCAATGATAAGAGC-3’.
[0014] Specifically, a PCR-amplified reaction system may include (25 pl): 0.25 pl of Taq Polymerase, 2.5 pl of 10xTaq Buffer, 2.5 ul of Mg ion Buffer, 2 ul of ANTPS, 0.5ul of an upstream primer PcanF (10 pM), 0.5 pul of a downstream primer PcanR (10 pM), 1pl of a genomic DNA, and 15.75 pl of ddH20.
[0015] The third object of the invention is to provide a method for assisting identification of P. canaliculata, wherein the method comprises the following steps: with the genomic DNA of the apple snail to be tested as a template, performing PCR amplification by using primers PcanF and PcanR, and determining whether the apple snail to be tested is P. canaliculata or not based on whether a PCR amplification product is obtained, wherein the sequences of the primers PcanF and PcanR are as follows:
[0016] PcanF (SEQ ID NO.1): 5’-GAGATGACCAGCTTTATAATGTC-3’;
[0017] PcanR (SEQ ID NO.2): 5’”-AAGAACCGGCAATGATAAGAGC-3°.
[0018] Further, the PCR amplification product has a size of 461 bp.
[0019] Specifically, the PCR amplification has the following reaction parameters: predegeneration at 94°C for 3 min, degeneration at 94°C for 3 min, annealing at 60°C for sec, extension at 72°C for 40 sec, 35 cycles, and extension at 72°C for 5 min.
[0020] A determination method according to the invention is as follows: after the LU101603 amplification product is electrophoretically imaged, the sample to be tested is determined to be the P. canaliculata if a band is shown from the PCR amplification product; and the sample to be tested is determined to be not the P. canaliculata if no band is obtained from the PCR amplification product. |
[0021] The invention has the following advantageous effects.
[0022] (1) The species identification process is rapid and efficient with accurate and reliable identification results. The kit of the specific primers specifically amplifies a product of a particular size with respect to the P. canaliculata, and is high in sensitivity, low in time consumption and capable of implementing high-throughput testing.
[0023] (2) Testing objects are not affected by gender, developmental stages, and tissue integrity.
[0024] (3) The identification process is simple, convenient, fast and accurate and is suitable for large-scale standardized operations, which is convenient for staff without professional taxonomy knowledge background.
[0025] FIG. 1 is gel electrophoresis of Pomacea canaliculata from different geographical populations as tested with a primer pair PcanF/PcanR.
[0026] FIG. 2 is gel electrophoresis of Pomacea canaliculata at different developmental stages as tested with a primer pair PcanF/PcanR.
[0027] FIG. 3 is gel electrophoresis of Pomacea canaliculata tested with a primer pair PcanF/PcanR in terms of sensitivity test.
[0028] The embodiments below further illustrate but do not limit the invention.
The experimental methods in the embodiments below are conventional methods, unless LU101603 otherwise stated. Test materials used in the embodiments below are purchased from biochemical reagent stores, unless otherwise stated. For quantitative tests in the embodiments below, three repeated experiments were set up, and the results were averaged.
[0029] Samples in the embodiments include P canaliculata and Pomacea maculata, both collected from different locations, both of which were publicly available.
[0030] Embodiment 1: Design of specific primer pair
[0031] On the basis of obtaining COI barcode segments of two types of invasive apple snails, the present invention, through multiple comparative analysis of sequences, designed and synthesized a specific primer pair capable of identifying the P. canaliculate as follows:
[0032] PcanF (SEQ ID NO.1): 5’-GAGATGACCAGCTTTATAATGTC-3’; Tm=55.30°C, GC%=39.13;
[0033] PcanR (SEQ ID NO.2): 5-AAGAACCGGCAATGATAAGAGC-3’; Tm=55.30°C, GC%=45.45.
[0034] Embodiment 2: Detection of P. canaliculata
[0035] In this embodiment, apple snails used were collected from the following geographical populations: samples 1-4: collected from Zhoushan, Zhejiang Province; samples 5-6: collected from Jieyang, Guangdong Province; samples 7-8: collected from Guangzhou, Guangdong Province; samples 9-10: collected from Yuyao, Zhejiang Province; Samples 11-12: collected from Nanning, Guangxi Province; samples 13-14: collected from Fuzhou, Fujian Province; and samples 15-16: collected from Changsha, Hunan Province. Among them, the samples 1-8 were Pomacea canaliculata, and samples 9-16 were not Pomacea canaliculata.
[0036] 1. Extraction of genomic DNAs (gDNAs): the gDNAs of single eggs were LU101603 extracted using the genomic DNA extraction kit from Tiangen Biotech (Beijing). There were 16 samples in total. Specifically, the gDNAs were extracted through the following steps.
[0037] (1) The surface of the single egg of each golden apple snail was washed with 75% alcohol, washed twice with distilled water, dried in air on a piece of clean filter paper, and then transferred to a 1.5 ml PCR tube.
[0038] (2) 180 pl of GA buffer was added to a centrifuge tube, and the shell of the egg was broken with a pipette tip to release tissue fluid in the egg.
[0039] (3) 20 pl of ProK (20 mg/ml) was added and then mixed by shaking, and then held in a water bath at 56°C for 1 h.
[0040] (4) 200 pl of buffer GB was added and then mixed by inverting thoroughly, and then held in a water bath at 70°C for 10 min, and when a solution became clear, the solution was centrifuge shortly.
[0041] (5) 200 pl of absolute ethanol was added and then mixed by shaking thoroughly, and centrifuging was conducted shortly.
[0042] (6) The solution was transferred to an adsorption column CB3, which was placed into a collection tube and then centrifuged at 12,000 rpm for 30 sec; waste liquid was discarded; and the adsorption column was placed into the collection tube.
[0043] (7) 500 pl of buffer GD was added to the adsorption column CB3, which was centrifuged at 12000 rpm for 30 sec; waste liquid was discarded; and the adsorption column was placed back to the collection tube.
[0044] (8) 600 ul buffer PW was added to the adsorption column CB3, which was centrifuged at 12000 rpm for 30 sec; waste liquid was discarded; and the adsorption column was placed back to the collection tube.
[0045] (9) Step (8) was repeated.
[0046] (10) The adsorption column CB3 was placed back into the collection tube LU101603 and centrifuged at 12000 rpm for 2 min; waste liquid was discarded; and the adsorption column CB3 was head at room temperature for several minutes, thereby completely drying a rinsing solution remaining in the adsorption material in air.
[0047] (11) The adsorption column CB3 was transferred to a clean centrifuge tube; 150 ul of buffer TE was dropwise added in the air to the adsorption column; and the adsorption column was held at room temperature for 2-5 min, and centrifuged at 12000 rpm for 2 min to obtain a genomic DNA solution.
[0048] 2. With the genomic DNAs from step 1 as templates, each genomic DNA was subject to PCR amplification by using the specific primer pair designed in Embodiment 1 to obtain a PCR amplification product.
[0049] A PCR reaction system included (25 pl): 0.25 pl of Taq Polymerase, 2.5 pl of 10xTaq Buffer, 2.5 ul of Mg?" Buffer, 2 pl of a dNTP misture, 0.5ul of the above upstream primer (10 uM), 0.5 pl of the above downstream primer (10 pM), ul of the genomic DNA, and 15.75 pl of ddH20.
[0050] PCR reaction conditions were as follows: predegeneration at 94°C for 3 min, degeneration at 94°C for 3 min, annealing at 60°C for 30 sec, extension at 72°C for 40 sec, 35 cycles, and extension at 72°C for 5 min.
[0051] 3. Testing of PCR products: 1 pl of each PCR amplification product from step 2 was subject to 1.5% agarose gel electrophoresis, stained with ethidium bromide (EB), and then observed in the gel system for imaging analysis.
[0052] The electrophoresis results of PCR amplifications of 16 samples with the specific primer pair PcanF/PcanR were shown in FIG.1. In FIG. 1, M indicated a DNA relative molecular weight standard (Marker I); and lanes 1 to 16 indicated samples 1 to 16 in sequence. In Fig. 1, the Pomacea canaliculata (Samples 1 to 8) collected from different locations all shown an amplified band, and other samples (Samples 9 to 16) shown noband. LU101603
[0053] 4. The PCR amplification products were recovered and sequenced respectively. The PCR amplification products of samples 1 to 8 were all sequenced as 461 bp. The sequencing results of samples 1-8 were shown in SEQ ID NO.3. After being compared with the GenBank database, the sequencing results shown that the sequences were 100% similar to the sequence of the published mitochondrial COI gene of the Pomacea canaliculata.
[0054] The results showed that by using the specific primer pair PcanF/PcanR, PCR products of specific length segments could be amplified from the P. canaliculata from different geographic populations, and no amplification band obtained from other species. The results were clear and easy to distinguish.
[0055] Embodiment 3: Testing samples of P canaliculata at different developmental stages
[0056] The P. canaliculata samples collected from Jieyang, Guangdong Province included the sample 17 which was an egg; a sample 18, which was young P. canaliculata that was three days old after being hatched; and a sample 19 which was adult P canaliculata.
[0057] 1. Extraction of gDNAs
[0058] The single young P canaliculata of the sample 18 was prepared by removing a shell carefully but with tissues therein remained. The single egg of the sample 17, the prepared P. canaliculata of the sample 18, and 100 g of pleopod tissues from the sample 19 were washed in 75% ethanol and washed twice with distilled water, dried in air on a piece of clean filter paper and then transferred to a 1.5 ml centrifuge tube, respectively. Other steps were the same as those in Embodiment 2.
[0059] 2. PCR amplification of P. canaliculata at different developmental stages by specific primers
[0060] With the gDNAs from step 1 as templates and sterile water as negative LU101603 control, each genomic DNA was subject to PCR amplification by using the specific primer pair designed in Embodiment 1 to obtain a PCR amplification product.
[0061] (1) PCR reaction system: same as Embodiment 2.
[0062] (2) Parameters of PCR reaction cycles: same as Embodiment 2.
[0063] (3) Testing of PCR products: same as Embodiment 2.
[0064] (4) The results of the specific primer pairs were shown in FIG. 2, where M indicated a DNA relative molecular weight standard (Marker I); and lanes 1 to 3 indicated samples 17 to 18 in sequence, and lane 4 indicated the negative control.
[0065] Embodiment 4: Sensitivity testing of specific primer pair for P. canaliculata
[0066] 1. Preparation of different gradient concentrations of template DNAs | [0067] Samples for the sensitivity testing of the specific primers included: the sample 1, which was P. canaliculata eggs collected from Zhoushan, Zhejiang Province.
[0068] The genomic DNAs were extracted through the same steps as those in Embodiment 2. The concentrations of the template DNAs were determined using NanoDrop. The template DNAs of the samples above were diluted to 0.1 ng/pl, 1 ng/pl, 5 ng/ul, 10 ng/pl, 25 ng/ul, 50 ng/pl, and 100 ng/pl in ddH2O, respectively. Through multiple repeated experiments and addition of the negative control, the sensitivity of each specific primer pair for detecting the P. canaliculata was determined according to the results.
[0069] 2. Sensitivity testing of specific primer pair for P. canaliculata
[0070] (1) PCR reaction system: same as Embodiment 2.
[0071] (2) Parameters of PCR reaction cycles: same as Embodiment 2.
[0072] (3) Testing of PCR products: same as Embodiment 2.
[0073] (4) The sensitivity testing results of respective primer pairs were shown in ee
FIG. 3, where M indicated a DNA relative molecular weight standard (D2000); 1 to 9 LU101603 indicated P. canaliculata template DNAs of 7 different concentrations of 0.1 ng/pl, 1 ng/ul, ng/ul, 10 ng/ul, 25 ng/pl, 50 ng/ul, and 100 ng/pl in sequence; and 8 indicated the negative control where the sterile water was used in place of the template DNA. The results showed that when the template concentration was 5 ng/pl, that is, when the 25 pl PCR system contained 5 ng of Pomacea canaliculate template DNA, weak bands could be amplified; and when more than 5 ng template DNAs was contained, bright bands could be amplified specifically. The primer pair has high sensitivity.
[0074] In summary, the results show that the specific primer according to the invention is accurate and reliable in idengifying the species of P. canaliculata, is applicable to samples at different developmental stages, and has the advantages of accuracy, sensitivity, convenience and rapidness.
Claims (4)
1. Primers for assisting identification of Pomacea canaliculata, wherein the primers refer to a primer pair PcanF and PcanR for amplifying a mitochondrial cytochrome oxidase subunit I gene of a golden apple snail, with sequences as follows: PcanF (SEQ ID NO.1): 5’-GAGATGACCAGCTTTATAATGTC-3’; PcanR (SEQ ID NO.2): 5”-AAGAACCGGCAATGATAAGAGC-3>.
2. À kit for assisting identification of Pomacea canaliculata, wherein the kit comprises a primer pair PcanF and PcanR for amplifying a mitochondrial cytochrome oxidase subunit I gene of a golden apple snail, with sequences as follows: PcanF (SEQ ID NO.1): 5”-GAGATGACCAGCTTTATAATGTC-3>; PcanR (SEQ ID NO.2): 5”-AAGAACCGGCAATGATAAGAGC-3>.
3. A method for assisting identification of Pomacea canaliculata, wherein the method comprises the following steps: performing PCR amplification by using primers PcanF and PcanR with a genomic DNA of a golden apple snail to be tested as a template, and determining whether the golden apple snail to be tested is a Pomacea canaliculata or not based on whether a PCR amplification product is obtained or not, wherein the sequences of the primers PcanF and PcanR are as follows: PcanF (SEQ ID NO.1): 5’-GAGATGACCAGCTTTATAATGTC-3>; PcanR (SEQ ID NO.2): 5”-AAGAACCGGCAATGATAAGAGC-3°.
4. The method according to claim 3, wherein the PCR amplification product has a size of 461 bp.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101603A LU101603B1 (en) | 2020-01-14 | 2020-01-14 | Primers, method and kit for assisting identification of Pomacea Canaliculata |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101603A LU101603B1 (en) | 2020-01-14 | 2020-01-14 | Primers, method and kit for assisting identification of Pomacea Canaliculata |
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| LU101603B1 true LU101603B1 (en) | 2020-05-18 |
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| LU101603A LU101603B1 (en) | 2020-01-14 | 2020-01-14 | Primers, method and kit for assisting identification of Pomacea Canaliculata |
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Effective date: 20200518 |