CN116949197A - Establishment of four common cryptosporidium RAA detection methods - Google Patents

Establishment of four common cryptosporidium RAA detection methods Download PDF

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CN116949197A
CN116949197A CN202311080419.8A CN202311080419A CN116949197A CN 116949197 A CN116949197 A CN 116949197A CN 202311080419 A CN202311080419 A CN 202311080419A CN 116949197 A CN116949197 A CN 116949197A
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cryptosporidium
raa
establishment
primer
parvum
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邓梦玲
贺君君
衡昭君
杨建发
邹丰才
舒凡帆
魏鹏浩
汪文雅
杨建岭
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Yunnan Agricultural University
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Yunnan Agricultural University
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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to establishment of four common cryptosporidium RAA detection methods. The RAA technology mainly uses 3 enzymes of recombinase, single-stranded DNA binding protein and DNA polymerase, under the condition of constant temperature, the recombinase and a specific primer are combined to form a complex, after the primer searches complementary sequences on template DNA, the template DNA is melted under the action of the single-stranded DNA binding protein, and then a new DNA complementary strand pair is formed under the action of the DNA polymerase. After repeated tens of times, the number of new DNA chains increases exponentially, and amplified fragments which can be detected by the device can be obtained within one hour, and the whole reaction is simple and rapid. The RAA technology has the advantages of convenience in operation, short reaction time, high sensitivity, convenience in carrying, good stability and the like, can be popularized and used in a basic layer or a remote mountain area, and has great application potential and prospect in field rapid diagnosis.

Description

Establishment of four common cryptosporidium RAA detection methods
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to establishment of four common cryptosporidium RAA detection methods.
Technical Field
At present, it has been confirmed that there are at least 44 effective species of Cryptosporidium, whose genotypes exceed 120, which can infect a wide variety of vertebrates including mammals, reptiles, amphibians, birds and fish. Cryptosporidiosis is considered to be a significant cause of diarrhea in humans and animals worldwide. Cryptosporidium can be transmitted through food and water, and human or animal infection with Cryptosporidium is mainly through ingestion of contaminated food or water, so that the incidence and prevalence of Cryptosporidium disease are high in underdeveloped and developing countries. Cases of human cryptosporidium infection are mostly children, symptoms such as malnutrition, recurrent vomiting, hypoevolutism, abdominal pain, diarrhea and the like can appear after infection, and serious cases even cause death. Cattle are recognized as a main source of cryptosporidiosis of people and livestock, the total infection rate of Cryptosporidiosis of cattle in China is 15.5 percent, and great public health hidden danger exists, and meanwhile, no effective therapeutic drug exists at present, so that a rapid, accurate, simple and convenient detection method is very important for preventing the cryptosporidiosis.
The current detection methods for cryptosporidium include: etiology diagnosis and immunology diagnosis. Microscopic examination of fresh fecal samples is the most straightforward way to detect the presence of cryptosporidium pathogen. The method combining dyeing with microscopic examination is developed subsequently to improve the detection sensitivity, however, the microscopic examination method has larger workload, not only has low sensitivity and specificity, but also cannot accurately identify the species and genotype of Cryptosporidium. Because of the large amount of impurities such as fecal slag, grease and the like in the feces, professional technicians are required to identify the feces when the cryptosporidium microscopic examination is carried out on the feces, and false negative or false positive can be caused due to insufficient recognition of oocyst morphology and impurities. Immunological diagnostic techniques have been developed as alternatives to microscopic detection, including enzyme-linked immunosorbent assays (ELISA), indirect ELISA, immunofluorescence techniques, and dipstick tests. The immunological technology has the advantages of higher sensitivity and specificity compared with microscopic examination, simpler operation and the like, but can only be used for detecting whether the cryptosporidium infection exists, the genotype of the cryptosporidium can not be determined, and the occurrence of false positive is easily caused by non-specific reaction.
With the rapid development of molecular biology techniques, detection methods based on PCR technology in combination with DNA sequencing have led to a number of new detection techniques for the identification of Cryptosporidium species, including Polymerase Chain Reaction (PCR), PCR restriction fragment length polymorphism analysis (PCR-RFLP), real-time fluorescent quantitative PCR, nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP), and the like. But the molecular biology method has the advantages of long time consumption, high requirements on instruments and equipment and technology, and difficulty in use and popularization in basic level and field diagnosis.
Disclosure of Invention
In order to solve the problems in the prior art, the invention adopts the following technical scheme:
the invention aims to provide a scheme for establishing RAA detection methods of four common Cryptosporidium.
It is another object of the present invention to provide the use of a method of RAA detection of Cryptosporidium in the detection of primary and on-site diagnosis of Cryptosporidium.
The Recombinase-mediated isothermal nucleic acid amplification (RAA) technology mainly uses 3 enzymes of Recombinase, single-stranded DNA binding protein (Single-stranded DNA binding, SSB) and DNA polymerase, wherein under the condition of constant temperature, the Recombinase and a specific primer are combined to form a complex, the primer searches complementary sequences on template DNA, the template DNA is melted under the action of the Single-stranded DNA binding protein, and a new DNA complementary strand pair is formed under the action of the DNA polymerase. After several tens of repetitions, the number of new DNA strands grows exponentially, and amplified fragments that can be detected by the device are obtained within one hour.
The invention has the beneficial effects that:
the whole reaction provided by the invention can react at normal temperature, is simple and quick, and has high sensitivity and strong specificity. The recombinant enzyme derived from bacteria or fungi used in the RAA technology is low in cost, and the interaction of various enzymes replaces the common PCR thermal cycle melting cycle process, so that the reaction time is greatly reduced, and the requirement on equipment is reduced.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.
FIG. 1 shows RAA amplification of Cryptosporidium angustifolium under different temperature conditions: m:2000bp DNA molecular mass standard; 1-7:36-42 ℃;
FIG. 2 shows RAA amplification of Cryptosporidium angustifolium under different amounts of primer conditions: m:2000bp DNA molecular mass standard; 1-6: 0.5-5. Mu.L;
FIG. 3 shows RAA amplification of Cryptosporidium angustifolium under varying amounts of magnesium acetate I: m:2000bp DNA molecular mass standard; 1-6: 1.5-4. Mu.L;
FIG. 4 shows the results of a Cryptosporidium angustifolium RAA plasmid susceptibility test: m:2000bp DNA molecular mass standard; 1-4:2.61×10 4 Copy/. Mu.L, 2.61X 10 3 Copy/. Mu.L, 2.61X 10 2 Copy/. Mu.L, 2.61X 10 1 Copy/. Mu.L;
FIG. 5 is a graph showing the specificity verification of Cryptosporidium angustifolium RAA: m:2000bp DNA molecular mass standard; 1-10: cryptosporidium angustiforme, cryptosporidium parvum, cryptosporidium nii, cryptosporidium parvum, pichia, giardia duodenum, toxoplasma, coccidium, capillary nematodes and roundworms;
FIG. 6 shows RAA amplification of Cryptosporidium parvum under different temperature conditions: m:2000bp DNA molecular mass standard; 1-7:36-42 ℃;
FIG. 7 shows RAA amplification of Cryptosporidium parvum under varying amounts of primer conditions: m:2000bp DNA molecular mass standard; 1-5: 0.5-4. Mu.L;
FIG. 8 shows RAA amplification of Cryptosporidium parvum under varying amounts of magnesium acetate I: m:2000bp DNA molecular mass standard; 1-5:1.5-3.5 mu L;
FIG. 9 shows the results of a Cryptosporidium parvum RAA plasmid susceptibility test: m:2000bp DNA molecular mass standard; 1-4:2.94×10 4 Copy/. Mu.L, 2.94X10 3 Copy/. Mu.L, 2.94X10 2 Copy/. Mu.L, 2.94X10 1 Copy/. Mu.L;
FIG. 10 shows the specificity verification of Cryptosporidium parvum RAA method: m:2000bp DNA molecular mass standard; 1-10: cryptosporidium parvum, cryptosporidium angustiforme, cryptosporidium nii, cryptosporidium parvum, pichia, giardia, toxoplasma, coccidium, capillary nematodes, roundworms;
FIG. 11 shows RAA amplification of Cryptosporidium bovis and Cryptosporidium ruihni under different temperature conditions: m:2000bp DNA molecular mass standard; 1-7:36-42 ℃; a: cryptosporidium bovis; b: cryptosporidium elegans;
FIG. 12 shows RAA amplification of Cryptosporidium bovis and Cryptosporidium ruihni under different amounts of primer conditions: m:2000bp DNA molecular mass standard; 1-5: 0.5-4. Mu.L; a: cryptosporidium bovis; b: cryptosporidium elegans;
FIG. 13 is RAA amplification of Cryptosporidium bovis and Cryptosporidium ruihni under varying amounts of magnesium acetate I: m:2000bp DNA molecular mass standard; 1-5:1.5-3.5 mu L; a: cryptosporidium bovis; b: cryptosporidium elegans;
FIG. 14 shows the results of experiments on the sensitivity of Cryptosporidium bovis and Cryptosporidium ruimachine RAA plasmids: m:2000bp DNA molecular mass standard; a: cryptosporidium bovis; 1-4:2.64×10 4 Copy/. Mu.L, 2.64X 10 3 Copy/. Mu.L, 2.64X 10 2 Copy/. Mu.L, 2.64X 10 1 Copy/. Mu.L; b: cryptosporidium elegans; 1-4: 2.24X10 4 Copy/. Mu.L, 2.24X10 3 Copy/. Mu.L, 2.24X10 2 Copy/. Mu.L, 2.24X10 1 Copy/. Mu.L;
FIG. 15 is a graph showing the specificity verification of Cryptosporidium bovis and Cryptosporidium ruimachine RAA: m:2000bp DNA molecular mass standard; a: cryptosporidium bovis; 1-10: cryptosporidium bovis, cryptosporidium angustifolium, cryptosporidium parvum, cryptosporidium ruthenicum, microsporum pichia, giardia duodenum, toxoplasma gondii, coccidium, capillary nematodes, roundworms; b: cryptosporidium elegans; 1-10: cryptosporidium elegans, cryptosporidium angustiforme, cryptosporidium parvum, cryptosporidium bovis, pichia, giardia duodenum, toxoplasma gondii, coccidium, capillary nematodes and roundworms;
FIG. 16 shows mRAA amplification of Cryptosporidium angustifolium and Cryptosporidium parvum under different temperature conditions: m:2000bp DNA molecular mass standard; 1-8:36-43 ℃;
FIG. 17 shows mRAA amplification of Cryptosporidium angustifolium and Cryptosporidium parvum primers in an amount of 1:1: m:2000bp DNA molecular mass standard; 1: the amount of the primer of the cryptosporidium angustiforme and the primer of the cryptosporidium parvum are 1 mu L;
FIG. 18 shows the amplification of mRAA by Cryptosporidium angustifolium and Cryptosporidium parvum different primers: m:2000bp DNA molecular mass standard; 1-7: cryptosporidium angustifolium: the usage amount of the cryptosporidium parvum primers is 0.5:1, 0.75:1, 1:1, 1:1.25, 1:1.5, 1:1.75 and 1:2 respectively;
FIG. 19 shows mRAA amplification of Cryptosporidium angustiforme and Cryptosporidium bovis under varying amounts of magnesium acetate I: m:2000bp DNA molecular mass standard; 1-5:1.5-3.5 mu L;
FIG. 20 shows the results of an experiment for sensitivity of Cryptosporidium angustifolium and Cryptosporidium parvum mRAA plasmids: m:2000bp DNA molecular mass standard; 1-4: the recombinant plasmid concentration was 10 in order 5 、10 4 、10 3 、10 2 Copy/. Mu.L amplification results;
FIG. 21 Cryptosporidium angustifolium and Cryptosporidium parvum RAA method specificity verification: m:2000bp DNA molecular mass standard; 1-12: cryptosporidium angustifolium and Cryptosporidium parvum mixture, cryptosporidium angustifolium, cryptosporidium parvum, cryptosporidium ruthenicum, cryptosporidium digitatum, giardia, coccidium, capillary nematodes and roundworms.
Description of the preferred embodiments
The present invention will be further described with reference to the following specific examples and drawings, but the present invention is not limited to the following examples.
In the following examples, the RAA reaction system was 50. Mu.L: 25 mu L of buffer solution, 0.5 to 5 mu L of forward and reverse primers (10 mu mol/L) and 1.5 to 4 mu L of DNA template (10) 1 ～10 4 Copy/. Mu.L), 1.5-4. Mu.L (280 mmol/L) of magnesium acetate I, and 50. Mu.L of distilled water, after being evenly mixed, the mixture is placed in a PCR instrument (or a water bath kettle) for constant-temperature amplification for 40min, and phenol/chloroform 1 is added: 1 mixture 50. Mu.L was mixed and 7. Mu.L was taken for gel electrophoresis identification.
Example 1
Establishment of cryptosporidium angustifolium RAA detection method
Optimization of the amplification conditions of Cryptosporidium angustifolium RAA:
primers were designed for the HSP70 gene locus of Cryptosporidium angustifolium (accession number: AB 089288), the upstream primer was 5 '-CCGTGCTTTGAGAAGATTAAGAACCCAGTG-3', and the downstream primer was 5 '-ACCTGCTGTTTCCAAACCTAAAGATAGTGG-3'. The reaction temperature, primers and amount of magnesium acetate I were varied to determine the optimal amplification conditions for RAA. The reaction components were added separately according to the reaction system, the reaction temperatures were set in the PCR apparatus at 36℃and 37℃and 38℃and 39℃and 40℃and 41℃and 42℃respectively, and the results of gel electrophoresis showed that the difference in the strip brightness was small at 36℃and 37℃and 38℃and that the strip at 40℃began to darken, as shown in FIG. 1, and 37℃was selected as the reaction temperature.
The primers were diluted to 10. Mu. Mol/L, the reaction temperature was 37℃and the concentrations of the primers (0.1. Mu. Mol/L, 0.2. Mu. Mol/L, 0.4. Mu. Mol/L, 0.6. Mu. Mol/L, 0.8. Mu. Mol/L) were varied, i.e., 0.5. Mu.L, 1. Mu.L, 2. Mu.L, 3. Mu.L, 4. Mu.L, 5. Mu.L of forward and reverse primers were added to the reaction system, respectively, and the gel electrophoresis showed that the bands became brighter as the amount of the primers increased, and that the 3. Mu.L and 4. Mu.L became darker as shown in FIG. 2, and the amount of the primers was 3. Mu.L (i.6. Mu. Mol/L, as the final concentration) was determined.
After adding 3. Mu.L of the primer at 37℃and the above reaction system, 1.5. Mu.L, 2. Mu.L, 2.5. Mu.L, 3. Mu.L, 3.5. Mu.L and 4. Mu.L of magnesium acetate I were added respectively, i.e., the final concentration of magnesium acetate was 8.4mmol/L,11.2mmol/L,14mmol/L,16.8mmol/L,19.6mmol/L and 22.4mmol/L, respectively, and the gel electrophoresis results showed that the bands of the amplified product became gradually brighter as the amount of magnesium acetate I increased, as shown in FIG. 3, the amount of magnesium acetate I was determined to be 3. Mu.L (i.e., the final concentration was 16.8 mmol/L).
Cryptosporidium angustifolium RAA sensitivity assay:
subjecting Cryptosporidium angustifolium plasmid standard to multiple dilution, and selecting concentration of 2.61×10 4 ～2.61×10 1 RAA amplification was performed using copy/. Mu.L as standard. As a result, as shown in FIG. 4, the RAA was able to detect at least 10 3 Copy/. Mu.L recombinant plasmid.
Cryptosporidium angustifolium RAA specificity experiments:
RAA amplification was performed using genomic DNA of Cryptosporidium angustiforme, cryptosporidium parvum, cryptosporidium Rugosae, pichia pastoris, giardia duodenum, toxoplasma gondii, coccidium, bursaphelenchus and ascariasis as templates, respectively, and the results are shown in FIG. 5: only when Cryptosporidium angustifolium DNA is used as a template, a single and brighter target band can be amplified, and other parasites do not amplify the band, so that the method has good specificity.
Example 2
Establishment of cryptosporidium parvum RAA detection method
Optimization of cryptosporidium parvum amplification conditions:
primers were designed for the cryptosporidium parvum SSU rRNA gene locus (accession number: MF 671870), with the upstream primer being 5 '-AAACTAATGCGAAAGCATTTGCCAAGGATG-3' and the downstream primer being 5 '-AAACTAATGCGAAAGCATTTGCCAAGGATG-3'. The reaction temperature, primers and amount of magnesium acetate I were varied to determine the optimal amplification conditions for RAA. The reaction temperature was set in the PCR apparatus at 36℃at 37℃at 38℃at 39℃at 40℃at 41℃at 42℃respectively, and the result of gel electrophoresis showed that the bands became gradually brighter as the temperature increased and that the bands became gradually darker when the temperature was higher to a certain level, and that the bands were brighter at 37℃at 38℃at 39℃as shown in FIG. 6, and that 37℃was selected as the reaction temperature.
The primers were diluted to 10. Mu. Mol/L, the reaction temperature was 37℃and the amount of the primers was determined to be 3. Mu.L (i.e., the final concentration was 0.6. Mu. Mol/L) at different primer concentrations (0.1. Mu. Mol/L, 0.2. Mu. Mol/L, 0.4. Mu. Mol/L, 0.6. Mu. Mol/L, 0.8. Mu. Mol/L) by adding 0.5. Mu. L,1. Mu. L, 2. Mu. L, 3. Mu. L, 4. Mu. L of forward and reverse primers to the reaction system, respectively, as shown in FIG. 7.
After the reaction system was completed at 37℃with 3. Mu.L of the primer, 1.5. Mu.L, 2. Mu.L, 2.5. Mu.L, 3. Mu.L, 3.5. Mu.L of magnesium acetate I was added, respectively, at a concentration of 8.4mmol/L,11.2mmol/L,14mmol/L,16.8mmol/L,19.6mmol/L,22.4mmol/L, and the gel electrophoresis showed that the band of the amplified product became brighter as the amount of magnesium acetate I increased, the band was brightest at 3. Mu.L of magnesium acetate I, and then darkened, as shown in FIG. 8, and the amount of magnesium acetate I was determined to be 3. Mu.L (i.e., the final concentration was 16.8 mmol/L).
Cryptosporidium parvum RAA sensitivity assay:
the Cryptosporidium parvum plasmid standard is subjected to multiple dilution, and the concentration is selected to be 2.94 multiplied by 10 4 ～2.94×10 1 RAA amplification was performed using copy/. Mu.L as standard. Gel electrophoresis results showed that RAA was detectable at least 10 2 Copy/. Mu.L recombinant plasmid as shown in FIG. 9.
Cryptosporidium parvum RAA specificity experiments:
RAA amplification was performed using Cryptosporidium angustifolium, cryptosporidium parvum, cryptosporidium ruticosae, pichia pastoris, giardia duodenum, toxoplasma gondii, coccidium, bursaphelenchus and ascarial genomic DNA as templates, respectively, as shown in FIG. 10: only when Cryptosporidium parvum is used as a template, a single and brighter target band can be amplified, and other target bands are not amplified, so that the method has good specificity.
Example 3
A single RAA detection method for Cryptosporidium bovis and Cryptosporidium ruier is established
The amplification conditions of Cryptosporidium bovis and Cryptosporidium ruier RAA are optimized:
primers are designed aiming at the HSP70 gene locus (accession number: AY 741306) of the cryptosporidium bovis, wherein the upstream primer is 5 '-GAAGTCAAGGCAACCGCAGGTGATACTCAC-3', and the downstream primer is 5 '-ATAAAGTGGCACGGAAGTAATCGGAACAAA-3'. Primers are designed aiming at HSP70 gene locus (accession number: MZ 787790) of Cryptosporidium ruisii, wherein the upstream primer is 5 '-AGAAAGAATCGCGGAATGGATTTGACTACA-3', and the downstream primer is 5 '-CTCTACTGGAGATAAAGTGGCACGGAAGT-3'. The reaction temperature, primers and amount of magnesium acetate I were varied to determine the optimal amplification conditions for RAA. The reaction temperature was set in the PCR apparatus to 36℃at 37℃at 38℃at 39℃at 40℃at 41℃at 42℃respectively, and electrophoresis was carried out in 2% gel after the completion of the reaction, which revealed that the Cryptosporidium bovis band was brighter at 37℃at 38℃at 39℃at 40℃and, when the temperature continued to rise, the band began to darken, as shown in FIG. 11A, and 37℃was selected as the reaction temperature. Cryptosporidium ruinanensis has little difference in band at different temperatures, and 37 ℃ is selected as a reaction temperature as shown in FIG. 11B.
The primer was diluted to 10. Mu. Mol/L, the reaction temperature was 37℃and forward and reverse primers of 0.5. Mu.L, 1. Mu.L, 2. Mu.L, 3. Mu.L and 4. Mu.L were added to the above reaction system, respectively, and the gel electrophoresis result showed that the bands of Cryptosporidium bovis and Cryptosporidium angustifolium became clear as the amount of the primers increased, and that the sizes and the brightness of the bands were appropriate when the amount of the primers was 3. Mu.L, as shown in FIG. 12, and the amount of the primers was 3. Mu.L (i.e., the final concentration was 0.6. Mu. Mol/L).
After 3. Mu.L of the primer was measured at 37℃and the reaction system was used, 1.5. Mu.L, 2. Mu.L, 2.5. Mu.L, 3. Mu.L and 3.5. Mu.L of magnesium acetate I were added, respectively, at a concentration of 8.4mmol/L,11.2mmol/L,14mmol/L,16.8mmol/L,19.6mmol/L and 22.4mmol/L, respectively, and the gel electrophoresis results showed that the amplified product bands of Cryptosporidium bovis and Cryptosporidium angustifolium anki were not significantly changed with the increase of the amount of magnesium acetate I, and the results were all brighter, and as shown in FIG. 13, the amounts of Cryptosporidium bovis and Cryptosporidium angustifolium anki were determined to be 3. Mu.L (i.8 mmol/L, final concentration).
Bovine and Cryptosporidium Russian RAA sensitivity experiments:
subjecting Cryptosporidium bovis and Cryptosporidium ruii plasmid standard to multiple ratio dilution, respectively, and selecting Cryptosporidium bovis plasmid concentration of 2.64×10 4 ～2.64×10 1 RAA amplification was performed using copy/. Mu.L as standard, and gel electrophoresis showed that RAA could be detected at least 10 3 Copyingmu.L plasmid as shown in FIG. 14A. Cryptosporidium ruishii susceptibility test the selected concentration was 2.64×10 4 ～2.64×10 1 RAA amplification was performed using copy/. Mu.L plasmid as standard, and gel electrophoresis showed that RAA could be detected at least 10 3 Copy/. Mu.L plasmid as shown in FIG. 14B.
Bovine and Cryptosporidium Russian RAA specificity experiments:
in the cryptosporidium bovis RAA specificity experiment, the cryptosporidium angustiforme, cryptosporidium parvum, cryptosporidium ruici, pichia, giardia duodenalis, toxoplasma, coccidium, capillary nematodes and ascarial genome DNA are used as templates for carrying out RAA amplification respectively, and the result is shown in figure 15A, and the result shows that: the target band can be amplified only when Cryptosporidium bovis is used as a template, and other parasites do not amplify the target band, which proves that the method has good specificity.
In the cryptosporidium ruihni RAA specificity experiment, the genomic DNA of cryptosporidium angustiforme, cryptosporidium parvum, cryptosporidium nii, cryptosporidium parvum, pichia, giardia duodenum, toxoplasma, coccidium, capillary nematodes and roundworms is used as a template to carry out RAA amplification, as shown in figure 15B: only when Cryptosporidium Rugosae DNA is used as a template, a target band can be amplified, and other parasites do not amplify the target band, so that the method has good specificity.
Example 4
The method for detecting the double RAA of the cryptosporidium angustiforme and the cryptosporidium parvum is established:
the double RAA amplification conditions of Cryptosporidium angustifolium and Cryptosporidium parvum are optimized:
cryptosporidium parvum and Cryptosporidium parvum RAA primers were determined according to examples 1 and 2, and the optimal amplification reaction conditions of the double RNA were explored from the reaction temperature, the primers, and the amount of magnesium acetate I, respectively. The reaction system was first subjected to an amplification reaction at 36, 37, 38, 39, 40, 41, 42, 43℃and the gel electrophoresis results showed that both bands were brighter at 37, 38℃and then the target band was gradually darkened with increasing temperature, as shown in FIG. 16, with 37℃being selected as the optimal reaction temperature for the subsequent experiments.
In the exploration of the primer dosage, the study firstly selects 1:1 of primer pair dosage, namely 1 mu L of cryptosporidium parvum and cryptosporidium angustifolium primer, and the result shows that the target strip of the cryptosporidium angustifolium in the amplified strip is brighter than the cryptosporidium parvum, as shown in figure 17, and then selects the reduction of the amount of the cryptosporidium angustifolium primer and the increase of the amount of the cryptosporidium parvum primer to explore the optimal primer ratio dosage. The forward and reverse primers were added to the above reaction system at a primer concentration of 10. Mu. Mol/L and a reaction temperature of 37℃respectively (0.5. Mu.L, 1. Mu.L), (0.75. Mu.L, 1. Mu.L), (1. Mu.L, 1.25. Mu.L), (1. Mu.L, 1.5. Mu.L), (1.75. Mu.L), (1. Mu.L, 2. Mu.L), and the gel electrophoresis results showed that the amplification was best when the amount of the Cryptosporidium angustifolium primer was 1. Mu.L (i.e., the final concentration was 0.2. Mu. Mol/L) and the amount of the Cryptosporidium parvum primer was 1.75. Mu.L (i.e., the final concentration was 0.35. Mu. Mol/L), as shown in FIG. 18.
After the reaction system was added, 1.5. Mu.L, 2. Mu.L, 2.5. Mu.L, 3. Mu.L and 3.5. Mu.L of magnesium acetate I were added, respectively, i.e., the magnesium acetate concentrations were 8.4mmol/L,11.2mmol/L,14mmol/L,16.8mmol/L and 19.6mmol/L, respectively, and the gel electrophoresis results showed that the difference in the strip brightness of the amplified product was not large with the increase in the amount of magnesium acetate I, as shown in FIG. 19, the amount of magnesium acetate I was determined to be 3. Mu.L (i.e., the final concentration was 16.8 mmol/L).
Dual RAA sensitivity experiments:
the Cryptosporidium angustifolium plasmid and the Cryptosporidium parvum plasmid standard are subjected to double dilution, and 1 mu L of each is added to perform double RAA amplification. As a result, gel electrophoresis showed that at least 10 could be detected 3 Copy/. Mu.L of Cryptosporidium plasmid and Cryptosporidium parvum plasmid, the results are shown in FIG. 20.
Dual RAA specificity experiments:
amplification was performed using the genomic DNA mixture of cryptosporidium angustiforme and cryptosporidium parvum, cryptosporidium angustiforme, cryptosporidium nieri, cryptosporidium parvum, pichia, giardia duodenalis, toxoplasma, coccidium, capillary nematodes, roundworms, respectively, as templates, and the results are shown in fig. 21: the mixture of the genomic DNA of the cryptosporidium angustiforme and the genomic DNA of the cryptosporidium parvum can amplify the bands of two items, the target bands of the cryptosporidium angustiforme and the genomic DNA of the cryptosporidium parvum can be respectively amplified when the genomic DNA of the cryptosporidium angustiforme and the genomic DNA of the cryptosporidium parvum are respectively used as templates, and the target bands of other parasites are not amplified, so that the method has better specificity.
While there have been described and shown what are at present considered to be the primary examples of the experimental factors, it will be understood by those skilled in the art that various changes, modifications and combinations can be made therein without departing from the spirit and scope of the invention as defined in the appended claims and their equivalents.

Claims (7)

1. The establishment of four common cryptosporidium RAA detection methods is characterized in that: the method comprises the establishment of a single RAA detection method of four kinds of Cryptosporidium angustiforme, cryptosporidium parvum, cryptosporidium bovis and Cryptosporidium ranunculus, and the establishment of a dual RAA detection method of the Cryptosporidium angustiforme and the Cryptosporidium parvum.
2. The establishment of the RAA detection method according to claim 1, comprising the steps of:
s1: designing and synthesizing a primer;
s2: optimizing RAA amplification conditions;
s3: RAA sensitivity experiments;
s4: RAA specificity experiments.
3. The establishment of the RAA detection method according to claim 2, wherein: primers for each cryptosporidium:
for cryptosporidium angustifolium:
an upstream primer: 5 '-CCGTGCTTTGAGAAGATTAAGAACCCAGTG-3'; SEQ ID NO.1;
a downstream primer: 5 '-ACCTGCTGTTTCCAAACCTAAAGATAGTGG-3'; SEQ ID NO.2;
for cryptosporidium parvum:
an upstream primer: 5 '-AAACTAATGCGAAAGCATTTGCCAAGGATG-3'; SEQ ID NO.3;
a downstream primer: 5 '-AAACTAATGCGAAAGCATTTGCCAAGGATG-3'; SEQ ID NO.4;
for Cryptosporidium bovis:
an upstream primer: 5 '-GAAGTCAAGGCAACCGCAGGTGATACTCAC-3'; SEQ ID NO.5;
a downstream primer: 5 '-ATAAAGTGGCACGGAAGTAATCGGAACAAA-3'; SEQ ID NO.6;
for Cryptosporidium ruishii:
an upstream primer: 5 '-AGAAAGAATCGCGGAATGGATTTGACTACA-3'; SEQ ID NO.7;
a downstream primer: 5 '-CTCTACTGGAGATAAAGTGGCACGGAAGT-3'. The method comprises the steps of carrying out a first treatment on the surface of the SEQ ID NO.8.
4. The establishment of the RAA detection method according to claim 1, wherein: the temperature after optimizing the conditions of the amplification system is selected to be 37 ℃, the addition amount of the primer is 3 mu L, the final concentration is 0.6 mu mol/L, 3 mu L of magnesium acetate I is taken, and the final concentration is 16.8mmol/L.
5. The establishment of the RAA detection method according to claim 1, wherein: plasmid concentration in the single RAA assay established sensitivity experiments was: cryptosporidium angustifolium 10 3 Multiple copies, cryptosporidium parvum 10 2 Multiple copies, cryptosporidium bovis 10 3 Multiple copies, cryptosporidium ruier 10 3 Copy number; plasmid concentration in the dual RAA assay established sensitivity experiments was: cryptosporidium angustifolium and Cryptosporidium parvum are both 10 3 And copies.
6. The establishment of the double RAA detection method for the cryptosporidium angustiforme and cryptosporidium parvum is characterized in that: cryptosporidium angustifolium and Cryptosporidium parvum are selected as the establishment of a dual RAA detection method for researching target sequences.
7. The method for dual RAA detection according to claim 6, wherein: primers in the single RAA detection method of the cryptosporidium angustiforme and the cryptosporidium parvum are selected, and the establishment of the double RAA detection method is carried out respectively from the experiments of optimizing amplification conditions, sensibility and specificity.
CN202311080419.8A 2023-08-25 2023-08-25 Establishment of four common cryptosporidium RAA detection methods Pending CN116949197A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117568501A (en) * 2024-01-15 2024-02-20 深圳市刚竹医疗科技有限公司 Primer probe combination, kit and method for detecting snake-source cryptosporidium based on real-time fluorescence quantitative PCR technology

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117568501A (en) * 2024-01-15 2024-02-20 深圳市刚竹医疗科技有限公司 Primer probe combination, kit and method for detecting snake-source cryptosporidium based on real-time fluorescence quantitative PCR technology

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