CPA kit, primers, applications and methods for detecting Eimeria coccidia and Cryptosporidium micrococcidia.

Abstract

This invention relates to the field of biodetection technology, specifically disclosing a CPA kit, primers, applications, and methods for detecting Eimeria coccidia and Cryptosporidium micrococcidia. The CPA kit for detecting Eimeria coccidia and Cryptosporidium micrococcidia of this invention includes the primer combinations shown in SEQ ID No. 1-12. Using the method of this invention, high sensitivity and high specificity detection of Eimeria coccidia and Cryptosporidium micrococcidia can be achieved within 15-45 minutes.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to a CPA kit, primers, applications, and methods for detecting Eimeria coccidia and Cryptosporidium micrococcidia. Background Technology

[0002] Eimeria is a common intestinal parasite in bovine animals, mainly causing bovine coccidiosis, which is characterized by acute enteritis and bloody stools. The infection rate in calves under 2 years old can be as high as 100%, and it has a high mortality rate, thus posing a serious threat to animal husbandry. Currently, 13 species of Eimeria have been identified in cattle: E. ellipsoidalis, E. subsperica, E. zuernii, E. ala bamensis, E. bovis, E. canadensis, E. cylindrica, E. auburnensis, E. wyomingensis, E. illinoisensis, E. brasiliensis, E. pellita, and E. bukidnonensis. Currently, *Eimeria tenella* and *Eimeria churnii* are widely considered to be the two most pathogenic *Eimeria tenella* species in cattle, causing intestinal damage, severe diarrhea, and even death. Therefore, rapid and easy-to-use detection methods are urgently needed to accurately identify the coccidioidomycete pathogen infecting cattle, which will contribute to precise prevention and control.

[0003] Cryptosporidium is a zoonotic protozoan that parasitizes the intestinal mucosal epithelial cells of humans and animals. It causes digestive disorders and diarrhea in the host and can be widely spread through water, food, and air, seriously threatening human and animal health. Studies have found that Cryptosporidium has the highest infection rate and intensity in dairy cows. Epidemiological surveys of Cryptosporidium show that the infection rate in pre-weaning calves is higher than that in other age groups within the same dairy farm. As the primary host for Cryptosporidium infection, dairy calves are mainly infected through feces and fecal-contaminated drinking water and feed. Calves housed in poorly cleaned and disinfected calf pens often lead to a large number of newborn calves becoming ill. Cryptosporidium infection severely damages the intestinal barrier and immune function. Furthermore, because Cryptosporidium oocysts are highly resistant to external environmental factors and resistant to many disinfectants, there are currently no effective drugs or vaccines for the prevention and control of Cryptosporidium disease.

[0004] Currently, the most commonly used method for detecting Eimeria coccidia and Cryptosporidium is quantitative real-time PCR. This method has good sensitivity and specificity, but it is complex to operate and the equipment is expensive, making it difficult to widely apply in actual production at the grassroots level. Therefore, research on its detection method is necessary. Summary of the Invention

[0005] The purpose of this invention is to provide a highly sensitive, specific, low-cost, and rapid method for detecting Eimeria coccidia and Cryptosporidium. Specifically, it provides a kit for the rapid and visual identification of pathogenic Eimeria coccidia and Cryptosporidium based on CPA cross-primer isothermal amplification technology, along with its dedicated primers and applications.

[0006] To achieve the objectives of this invention, the technical solution is as follows:

[0007] In a first aspect, the present invention provides a CPA kit for detecting Eimeria coccidia and Cryptosporidium micrococcidia, comprising the primer combinations shown in SEQ ID No. 1-12.

[0008] This invention designs CPA primer sets for detecting Eimeria based on the specific conserved target sequence 18S rRNA gene of Eimeria, and CPA primer sets for detecting Cryptosporidium based on the specific conserved target sequence cgd6_3910 gene of Cryptosporidium. The detection sensitivity for Eimeria can reach 5 copies / μl, and the detection sensitivity for Cryptosporidium can reach 10 copies / μl. It can detect both Eimeria and Cryptosporidium with high sensitivity, and the CPA detection method does not require expensive instruments, making it suitable for screening and detecting Eimeria and Cryptosporidium in primary healthcare units and disease control centers.

[0009] CPA-specific primers for detecting Eimeria coccidia (primer combination 1) include a stripping primer pair, a cross primer pair, and a detection primer pair, wherein:

[0010] The stripping primer pair includes F1 and B1, wherein the nucleotide sequence of F1 is shown in SEQ ID No. 1 and the nucleotide sequence of B1 is shown in SEQ ID No. 2;

[0011] The cross primer pair includes CPF1 and CPR1, wherein the nucleotide sequence of CPF1 is shown in SEQ ID No. 3 and the nucleotide sequence of CPR1 is shown in SEQ ID No. 4;

[0012] The detection primer pair includes QF1 and QR1, wherein the nucleotide sequence of QF1 is shown in SEQ ID No. 5 and the nucleotide sequence of QR1 is shown in SEQ ID No. 6.

[0013] CPA-specific primers for detecting Cryptosporidium globosum (primer combination 2) include a stripping primer pair, a cross primer pair, and a detection primer pair, wherein:

[0014] The stripping primer pair includes F2 and B2, wherein the nucleotide sequence of F2 is shown in SEQ ID No. 7 and the nucleotide sequence of B2 is shown in SEQ ID No. 8;

[0015] The cross primer pair includes CPF2 and CPR2, wherein the nucleotide sequence of CPF2 is shown in SEQ ID No. 9 and the nucleotide sequence of CPR2 is shown in SEQ ID No. 10;

[0016] The detection primer pair includes QF2 and QR2, wherein the nucleotide sequence of QF2 is shown in SEQ ID No. 11 and the nucleotide sequence of QR2 is shown in SEQ ID No. 12.

[0017] This invention selected several specific fragments from conserved sequences and obtained primer sequences capable of simultaneously detecting Eimeria coccidia and Cryptosporidium through extensive experimental replication and verification. Furthermore, in CPA detection, only the detection primers can be designed with software assistance; the other cross primers and stripping primers require extensive experimental and empirical verification to obtain.

[0018] The CPA kit of the present invention also includes 2.5×pH Sensitive Reaction Buffer and Bst II DNA Polymerase;

[0019] And / or, it also includes nucleic acid dyes and indicator dyes, said nucleic acid dyes including GelStain Blue or EqualbitdsDNA HS Reagent, said indicator dyes including OG Orange or Neutral Red.

[0020] The CPA kit of the present invention also includes a negative control, wherein the negative control is a system that does not contain Eimeria coccidia nucleic acid and Cryptosporidium globosum nucleic acid;

[0021] And / or, it also includes positive controls, wherein the positive controls are plasmids constructed by linking the 18S rRNA gene of Eimeria aureus and the cgd6_3910 gene of Cryptosporidium coccidioides to the pUC57 vector.

[0022] In a second aspect, the present invention provides primer combinations for detecting Eimeria coccidia and Cryptosporidium micrococcidia, the primer combinations comprising sequences as shown in SEQ ID No. 1-12.

[0023] Thirdly, the present invention provides the application of the primer combination in the preparation of reagents or kits for detecting Eimeria coccidia and Cryptosporidium micrococcidia.

[0024] Fourthly, the present invention provides a reagent containing the above-described primer combination.

[0025] Fifthly, the present invention provides a CPA method for detecting Eimeria coccidia and Cryptosporidium micrococcidia for non-disease diagnostic purposes, which uses the DNA of the sample to be tested as a template and performs cross-priming isothermal amplification using the above-mentioned CPA kit or primer combination or reagent.

[0026] In the CPA method of this invention, the reaction system for isothermal amplification of the cross-primers, in 25 μL volumes, comprises the following components at final concentrations: 1×pH Sensitive Reaction Buffer, 0.03-0.08 mmol / L indicator dye, 0.8-2 μmol / LF, 0.8-2 μmol / LB, 3.2-4 μmol / L CPF, 3.2-4 μmol / L CPR, 1.6-2 μmol / L QF, 1.6-2 μmol / L QR, and 600-700 units / mL Bst II DNA Polymerase; the DNA content is not less than 1 ng.

[0027] Wherein, F and B are stripping primer pairs, F includes F1 and F2, the sequence of F1 is shown in SEQ ID No. 1, the sequence of F2 is shown in SEQ ID No. 7, and B includes B1 and B2, the sequence of B1 is shown in SEQ ID No. 2, and the sequence of B2 is shown in SEQ ID No. 8; preferably, the concentrations of F1 and F2 are the same, and the concentrations of B1 and B2 are the same;

[0028] CPF and CPR are cross-primer pairs. CPF includes CPF1 and CPF2, the sequence of CPF1 is shown in SEQ ID No. 3, and the sequence of CPF2 is shown in SEQ ID No. 9. CPR includes CPR1 and CPR2, the sequence of CPR1 is shown in SEQ ID No. 4, and the sequence of CPR2 is shown in SEQ ID No. 10. Preferably, the concentrations of CPF1 and CPF2 are the same, and the concentrations of CPR1 and CPR2 are the same.

[0029] QF and QR are detection primer pairs. QF includes QF1 and QF2, the sequence of QF1 is shown in SEQ ID No. 5, and the sequence of QF2 is shown in SEQ ID No. 11. QR includes QR1 and QR2, the sequence of QR1 is shown in SEQ ID No. 6, and the sequence of QR2 is shown in SEQ ID No. 12. Preferably, the concentrations of QF1 and QF2 are the same, and the concentrations of QR1 and QR2 are the same.

[0030] And / or, after the reaction has proceeded, a step of adding nucleic acid dye may also be included.

[0031] In the CPA method of the present invention, the reaction conditions for the isothermal amplification of the cross primers include: a reaction temperature of 60-65°C, preferably 63°C, and a reaction time of 15-45 min.

[0032] And / or, the samples to be tested include animal feed, cattle and sheep products, raw materials for animal vaccine production, or semi-finished animal vaccines.

[0033] This invention can be used to detect Eimeria coccidia and Cryptosporidium in the environment and industrial products (such as meat products, feed, vaccine-related products, etc.) to monitor environmental safety and product quality.

[0034] In the CPA method of the present invention, if the CPA amplification product of the test sample has a clear target band in the gel electrophoresis result compared with the negative control, and / or has obvious fluorescence under ultraviolet / blue light, and / or has obvious color change reaction observed by the naked eye, then the test sample is determined to be positive; otherwise, it is determined to be negative.

[0035] When the sample to be tested is determined to be positive, it can be further tested with primer combination 1 or 2 of the present invention to further confirm whether it is Eimeria coccidia positive or Cryptosporidium micrococcus positive.

[0036] As a specific embodiment, the detection method of the present invention includes:

[0037] 1) Extract nucleic acid from the sample to be tested (phenol-chloroform extraction or commercial extraction kits are recommended);

[0038] 2) Using the primer combination or CPA kit described above and the DNA extracted in step 1) or negative control, prepare a CPA reaction system and perform cross-primer isothermal amplification;

[0039] 3) Make a judgment based on the results of the cross-primer isothermal amplification in step 2).

[0040] The beneficial effects of this invention are at least as follows:

[0041] The detection method and kit for Eimeria coccidia and Cryptosporidium provided by this invention offer high sensitivity (the detection limit for Eimeria coccidia is 5 copies / μl, and the detection limit for Cryptosporidium is 10 copies / μl) and high specificity for detecting these two parasites. Furthermore, it features a short detection cycle (detection can be completed within 15-45 minutes, with high efficiency), isothermal detection, low equipment requirements (no complex instruments are needed), low skill requirements for identification and testing personnel, and visualized detection results. Therefore, the CPA-specific primers and kit provided by this invention are suitable for rapid on-site detection of early Eimeria coccidia and Cryptosporidium outbreaks, enabling timely control of the epidemic. Attached Figure Description

[0042] Figure 1 The results of gel electrophoresis comparing the specificity of each pair of primer sets 1 and 2 with primer sets 3 and 4 in Example 1 are as follows:

[0043] Figure 2 The images show gel electrophoresis images of the products obtained by amplifying the DNA of Eimeria coccidia and Cryptosporidium micrococcidia under different reaction temperature conditions in Example 2 of this invention using the CPA primer sets No. 1 and No. 4 provided by this invention.

[0044] Figure 3 The images show gel electrophoresis results of the products obtained by co-amplifying the DNA of Eimeria coccidia and Cryptosporidium microsporidium using the CPA primer sets No. 1 and No. 4 provided by the present invention under different reaction time conditions in Example 2 of the present invention.

[0045] Figure 4 This is a gel electrophoresis image showing the specific detection results of Eimeria coccidia and Cryptosporidium micrococcidia using the CPA primer set in Example 3 of this invention.

[0046] Figure 5 This is an ultraviolet image showing the results of the specific detection of Eimeria coccidia and Cryptosporidium micrococcidia using the CPA primer set in Example 3 of this invention.

[0047] Figure 6 This is a photograph showing the orange-yellow discoloration of the results of the specific detection of Eimeria coccidia and Cryptosporidium micrococci in Example 3 of this invention by the CPA primer set;

[0048] Figure 7 This is a gel electrophoresis image showing the sensitivity detection results of the CPA primer set for Eimeria coccidia and Cryptosporidium micrococci in Example 3 of the present invention.

[0049] Figure 8 The image shown is an ultraviolet light image of the sensitivity detection results of the CPA primer set for Eimeria coccidia and Cryptosporidium micrococci in Example 3 of this invention.

[0050] Figure 9 This is a photograph showing the orange-yellow color change of the CPA primer set in Example 3 of the present invention, which is a visual observation of the sensitivity detection results of Eimeria coccidia and Cryptosporidium micrococci.

[0051] Figure 10 These are images of the orange-yellow color change observed by the naked eye of five clinical samples in Example 5 of this invention.

[0052] Figure 11 Images of five clinical samples from Example 5 of this invention under ultraviolet light. Detailed Implementation

[0053] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The microorganisms used in this invention can be obtained based on common technical knowledge in the field or are commercially available. For example, bovine rotavirus (BRV) is disclosed in Jin Yuhang. Isolation and Identification of Bovine Rotavirus HB01 Strain and Preliminary Study on VP7 Subunit Vaccine [D]. Hebei University of Science and Technology, 2022. DOI:10.27741 / d.cnki.ghbkj.2022.000075.; Bovine Infectious Rhinotracheitis Virus (IBRV) and Bovine Viral Diarrhea Virus (BVDV) are disclosed in Zhao Zhuo, Hu Yibin, Lei Lihui, Wu Peize, Wu Chunxia, ​​Ma Yan, Zhang Jufeng, Wang Li, Jiang Housheng. Trivalent Bovine Viral Diarrhea / Mucosal Disease (Type 1 + Type 2), Bovine Infectious Rhinotracheitis, and Bovine Parainfluenza (Type 3). Development and evaluation of the immunization effect of inactivated vaccine (E2 protein + C1 strain + HB01 strain) [J]. Chinese Journal of Veterinary Drugs, 2022, 56(11):17-22.; Bovine Mycoplasma (PPLO) is disclosed in Wu Zhiqiang, Shi Yuemeng, Kou Zhaoting, Wang Xin, Huang Suwen, Duan Yueqiang. Isolation and identification of bovine respiratory disease pathogens BVDV, IBRV, BPIV3 and Bovine Mycoplasma [J]. Contemporary Animal Husbandry, 2022(05):12-16. DOI:10.14070 / j.cnki.15-1150.2022.05.004.; Bovine hemolytic Manslaughter bacillus (Mh) is disclosed in Chinese Patent CN202110355541.6.

[0055] For detailed steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., 3rd edition, 2001, NY, Cold Spring Harbor).

[0056] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials in this invention. In fact, the sources of the biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments. All sequences involved in the embodiments were synthesized using existing technologies.

[0057] Example 1: Determination of CPA primers for differentiating pathogenic Eimeria auburnensis from Cryptosporidium parvum

[0058] 1.1 Design of CPA primers for identifying pathogenic Eimeria auburnensis and Cryptosporidium parvum:

[0059] In this embodiment, the inventors designed primers using the conserved region of the 18S rRNA gene of *Eimeria tenella* and the cgd6_3910 gene of *Cryptospora microsporidium*, using the primer design software Primer Premier. 5. Based on the following primer design principles, design CPA primers for detecting Eimeria coccidia by adjusting parameters such as Tm value, GC content, dG critical value, amplification length, and fragment region: (1) Adjust the Tm value to 60-65℃ to reduce false positive results; (2) Adjust the length of CPFc / CPRc primers to 20-22bp and the length of F / B primers to 18-20bp; (3) To control the specific occurrence of the reaction, set the ΔG values ​​of the 5' and 3' ends of CPFc and CPRc primers and the ΔG values ​​of F / B primers to be less than -4Kcal / mol, and set dimercheck to -2; (4) Adjust the GC content to 40-60% to improve product specificity and yield; (5) Based on the above parameter settings, screen the optimal combination of stripping primer and cross primer sequences through experiments.

[0060] After screening two sets of stripping primers and cross primers based on the above experimental process, the selected stripping primers and cross primers were first subjected to specificity verification. After confirming that the negative control had no band, detection primers were designed according to the above parameters. Sensitivity detection experiments were conducted on the detection primers to screen out the optimal detection primers. Sensitivity is reflected by the limit of detection, which is the lowest detection concentration at which the lowest amount of template added can be detected, and the product shows a band, orange-yellow color change, or green fluorescence. Based on the above primer design principles, multiple sets of CPA primer combinations were designed. Table 1 below only lists two sets of primers for Eimeria coccidia and Cryptosporidium globosum (primer sets 1 and 2, and primer sets 3 and 4). From these, CPA primer sets with high specificity that can be used to identify and detect pathogenic Eimeria coccidia and Cryptosporidium globosum were determined.

[0061] Table 1. CPA primer set for differentiating pathogenic Eimeria coccidia and Cryptosporidium coccidioides.

[0062]

[0063]

[0064] 1.2 Screening of CPA primers for differentiating pathogenic Eimeria coccidia and Cryptosporidium micrococcidia

[0065] Two sets of CPA primers for *Eimeria tenella* (primer set 1 and primer set 2) and two sets of CPA primers for *Cryptospora microsporidium* (primer set 3 and primer set 4) from Table 1 were used in pairs to verify the specificity of the primer combinations. CPA amplification was performed on a negative control (nuclease-free distilled water) to select the optimal CPA primer combination. Specifically, 25 μl of the reaction system for CPA amplification detection was added to a 0.2 ml EP reaction tube. Specific information about the reaction system is shown in Table 2 (in this embodiment, the DNA sample in Table 2 is not added during primer screening; this part is replaced by a negative control). After the reaction system was prepared, it was placed in a conventional PCR instrument and reacted at 65°C for 60 min. The amplification products were then detected by gel electrophoresis. The results are shown in Table 2. Figure 1 As can be seen from this, primer set 1 and primer set 4 have higher specificity and no non-specific amplification results. Therefore, this invention determines primer set 1 as the best CPA primer for detecting Eimeria coccidia and primer set 4 as the best CPA primer for detecting Cryptosporidium globosum.

[0066] Table 2 CPA amplification reaction system

[0067]

[0068] Example 2

[0069] The 18S rRNA gene of Eimeria coccidia (AB769563.1) and the cgd6_3910 gene of Cryptosporidium globosum (XM_627728.1) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The two synthesized genes were ligated into the pUC57 plasmid (with SacI / SalI restriction sites) and the recombinant vector DNA was used as a positive plasmid.

[0070] 2.1 Determination of amplification time

[0071] This embodiment utilizes the combination of primer set 1 and primer set 4 determined in Example 1 to perform CPA amplification detection on positive plasmids extracted from *Eimeria tenella* and *Cryptospora microspora*. Specifically, multiple CPA amplification reaction systems were prepared according to Table 2 above. After preparation, all systems were placed on a standard PCR instrument for CPA amplification. The amplification reaction program was: 65℃, 60 min, 45 min, 35 min, 25 min, and 15 min, with a negative control (-). The amplification products at different amplification times were detected by gel electrophoresis, and the results are shown below. Figure 3 As shown.

[0072] according to Figure 3 The results show that positive DNA samples of Eimeria coccidia and Cryptosporidium can be detected under amplification time of 15 min or more, and the bands detected by gel electrophoresis are clear. Therefore, the CPA primer set provided by this invention for identifying pathogenic Eimeria coccidia and Cryptosporidium can detect results within 15-60 min, achieving the purpose of rapid detection.

[0073] 2.2 Determination of amplification temperature

[0074] This embodiment utilizes primer sets 1 and 4, determined in Example 1, to perform CPA amplification detection on DNA samples extracted from *Eimeria tenella* and *Cryptospora microsporidium*, respectively (i.e., primer set 1 and *Eimeria tenella* DNA were detected separately, and primer set 4 and *Cryptospora microsporidium* DNA were detected separately). The specific procedure is as follows: multiple CPA amplification reaction systems were prepared according to Table 2 above (the primers in primer set 4 were not included when detecting *Eimeria tenella* DNA samples, and the primers in primer set 1 were not included when detecting *Cryptospora microsporidium* DNA samples). After the amplification reaction systems were prepared, they were all placed on a conventional PCR instrument for CPA amplification. The amplification reaction program was: 25℃, 60℃, 65℃, 45 min, with a negative control (-). The amplification products under different amplification temperature conditions were detected by gel electrophoresis, and the results are as follows. Figure 2 As shown.

[0075] according to Figure 2The results show that the amplification effect (brightest band) is best at 60℃ when detecting Eimeria coccidia, followed by 65℃. For Cryptosporidium micrococcus, 65℃ is optimal. Considering all factors, this invention determines 60-65℃ as the amplification temperature for the CPA primer set for identifying pathogenic Eimeria coccidia, preferably 63℃.

[0076] Example 3: Verification of CPA primers for differentiating pathogenic Eimeria coccidia and Cryptosporidium micrococcidia.

[0077] 3.1 Specificity Validation

[0078] (1) Virus strain and sample

[0079] Following the QIAGEN instructions, positive plasmids (+) for bovine rotavirus (BRV), bovine infectious rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), bovine mycoplasma (PPLO), Escherichia coli (E. coli), bovine hemolytic Mansonia (Mh), bovine Eimeria auburnensis, and bovine Cryptosporidium parvum were extracted as test samples, and negative controls (-) were set up.

[0080] (2) CPA amplification reaction systems for different DNA samples were prepared according to the CPA amplification reaction systems shown in Table 2 above. OG orange (added before the reaction, concentration 0.05 mmol / L) and Equalbit dsDNA HS Reagent nucleic acid dye (added after the reaction, concentration 0.05 mmol / L) were added to the amplification reaction systems. After preparation, all amplification reaction systems were placed on a conventional PCR instrument for CPA amplification. The amplification reaction program was 63℃ for 120 min. The amplification products for different DNA samples were detected by gel electrophoresis. The results are shown below. Figure 4 As shown; the results of observing each amplification tube under ultraviolet light are as follows. Figure 5 As shown; each amplification tube was visually observed, and the magenta-orange-yellow color change results are as follows. Figure 6 As shown.

[0081] according to Figure 4 The results show that only positive plasmids for Eimeria auburnensis and Cryptosporidium parvum showed amplification bands; according to Figure 5The results show that only the amplification products of plasmids positive for Eimeria auburnensis and Cryptosporidium parvum exhibited significant green fluorescence under ultraviolet light; according to Figure 6 The results show that only the amplification products of positive plasmids targeting *Eimeria auburnensis* and *Cryptosporidium parvum* exhibited a visual orange-yellow discoloration. These results indicate that within 120 minutes of amplification, only positive plasmids targeting *Eimeria auburnensis* and *Cryptosporidium parvum* were detected, while six other pathogens similar to *Eimeria auburnensis* or causing similar symptoms were not detected. This demonstrates that the CPA primer set of this invention for identifying and detecting pathogenic *Eimeria auburnensis* and *Cryptosporidium parvum* has good specificity.

[0082] 3.2 Sensitivity Verification

[0083] This embodiment utilizes primer set 1 and primer set 4 determined in Example 1 above to target different concentrations (Eimeria auburnensis 18S rRNA genome and Cryptosporidium parvumcgd 6_3910 genome standard positive plasmid (5 8 copy / μl and 10 8 (copy / μl) was obtained by serial dilution at 5-fold and 10-fold respectively. 8 With 10 8 copy / μl, 5 7 With 10 7 copy / μl, 5 6 With 10 6 copy / μl, 5 5 With 10 5 copy / μl, 5 4 With 10 4 copy / μl, 5 3 With 10 3 copy / μl, 5 2 With 10 2 copy / μl, 5 1 With 10 1 The sample size was measured at 1000 μl to evaluate the sensitivity of the CPA primers provided by this invention for detecting Eimeria coccidia and Cryptosporidium micrococcidia.

[0084] The 25 μl reaction system is shown in Table 2 above (with OG orange added to the reaction system at a concentration of 0.05 mmol / L, and EqualbitdsDNA HS Reagent nucleic acid dye added after the reaction at a concentration of 0.05 mmol / L). The reaction conditions were 63℃ for 45 min. The results of gel electrophoresis detection of the amplification products for DNA samples of different concentrations are shown below. Figure 7 As shown; the results of observing each amplification tube under ultraviolet light are as follows. Figure 8 As shown; each amplification tube was visually observed, and the magenta-orange-yellow color change results are as follows. Figure 9 As shown. Figure 7 , Figure 8 , Figure 9 Primer set 1 represents the concentration of Eimeria coccidia positive plasmid, and primer set 4 represents the concentration of Cryptosporidium micrococcus positive plasmid.

[0085] according to Figure 7 The results show that bands can still be detected even when the positive plasmids *Eimeria auburnensis* 18S rRNA are at 5 copies / μl and *Cryptosporidium parvum* cgd 6_3910 are at 10 copies / μl; according to Figure 8 The results show that the DNA content in the sample was 5 copies / μl for Eimeria auburnensis 18S rRNA and 10 copies / μl for Cryptosporidium parvum cgd 6_3910. The amplified products still exhibited significant green fluorescence under ultraviolet light. According to... Figure 9 The results show that the concentration of DNA in the sample was 5 copies / μl for Eimeria auburnensis 18S rRNA and 10 copies / μl for Cryptosporidium parvum cgd 6_3910. The amplification products showed a magenta-orange color change when observed with the naked eye.

[0086] Example 4: CPA kit for detecting Eimeria coccidia and Cryptosporidium micrococcidia.

[0087] This embodiment provides a CPA kit for detecting Eimeria coccidia and Cryptosporidium glomeratum, comprising the CPA primer set (primer set 1) for detecting Eimeria coccidia and the CPA primer set (primer set 4) for detecting Cryptosporidium glomeratum determined in Example 1 above. It also includes nucleic acid dyes and enzyme reaction solutions for preparing the reaction system for isothermal amplification of cross-primers to detect Eimeria coccidia and Cryptosporidium glomeratum. The nucleic acid dyes include EqualbitdsDNA HS Reagent (added after the reaction), and the enzyme reaction solution includes the following components: 2.5×pH Sensitive Reaction Buffer (purchased from YEASEN), and OG orange (added before the reaction). For ease of reaction system preparation, the CPA kit also includes a negative control (e.g., nuclease-free, nucleic acid-free distilled water) and a positive control (e.g., plasmids containing Eimeria coccidia 18S rRNA and Cryptosporidium glomeratum cgd6_3910 gene DNA).

[0088] To facilitate the use of the kit provided in this embodiment, the kit also includes a cross-primer isothermal amplification method for detecting Eimeria coccidia and Cryptosporidium micrococcidia, which may include the following steps:

[0089] 1) Extract DNA from the sample to be tested (phenol-chloroform extraction or commercial extraction kits are recommended);

[0090] 2) The DNA sample extracted in step 1) was subjected to cross-priming isothermal amplification using the kit provided in this embodiment; wherein the 25 μL reaction system for cross-priming isothermal amplification comprises the following components at final concentrations: 1× pHSensitiveReaction Buffer, 0.03-0.08 mmol / L indicator dye, 0.4-1 μmol / L F1, 0.4-1 μmol / L LF2, 0.4-1 μmol / L B1, 0.4-1 μmol / L B2, 1.6-2.0 μmol / L CPF1, 1.6-2.0 μmol / L CPF2, 1.6-2.0 μmol / L CPR1, 1.6-2.0 μmol / L CPR2, 0.8-1.0 μmol / L QF1, 0.8-1.0 μmol / L QF2, 0.8-1.0 μmol / L QR1, 0.8-1.0 μmol / L QR2 and 600-700 units / mL Bst II DNA Polymerase; the isothermal amplification reaction conditions for cross-priming are: reaction temperature 60-65℃, preferably 63℃, reaction time 15-45 min. After the reaction, nucleic acid dye is added at a concentration of 0.03-0.08 mmol / L in the system.

[0091] 3) Determine the outcome based on the amplification results from step 2):

[0092] If the negative control does not show obvious green fluorescence or orange-yellow discoloration, and the negative control shows no obvious bands on agarose gel electrophoresis, the test result is accurate and reliable. If the test sample shows obvious green fluorescence under ultraviolet light or orange-yellow discoloration upon visual inspection, or shows obvious bands on agarose gel electrophoresis, it is judged as positive. If there is no obvious green fluorescence, orange-yellow discoloration, or obvious bands on agarose gel electrophoresis, it is judged as negative.

[0093] If the negative control also shows obvious green fluorescence or orange-yellow discoloration, and the agarose gel electrophoresis experiment shows obvious bands, it indicates that the reagent is contaminated and the experiment should be repeated or a new reagent should be used for testing. If the positive control does not change after adding nucleic acid dye, it indicates that the reagent is invalid and the experiment should be repeated or a new reagent should be used for testing.

[0094] Example 5: Clinical Sample Testing

[0095] In this embodiment, the reagent kit provided in Example 4 above was used to perform CPA qualitative detection on five clinical bovine tissue DNA samples provided by the Animal Routine Diseases and Pathogens Research Center of Jinyu Baoling Biological Pharmaceutical Co., Ltd., according to the detection method steps provided in the reagent kit.

[0096] Sample preparation method: Bovine tissue was added to three times the volume of PBS, along with a small amount of steel balls and quartz sand, and then the tissue was broken up in a homogenizer for 15 minutes. 400 μL of the supernatant was used for DNA extraction, and 2 μL of the extracted DNA was taken for testing.

[0097] In the 25 μL reaction system, the concentrations of F1, F2, B1, and B2 were all 0.4 μmol / L; the concentrations of CPF1, CPF2, CPR1, and CPR2 were all 1.6 μmol / L; the concentrations of QF1, QF2, QR1, and QR2 were all 0.8 μmol / L; and the concentration of Bst II DNA Polymerase was 600 units / mL. The enzyme reaction solution was 1×pH Sensitive Reaction Buffer (12.5 μL, containing OG Orange at a concentration of 0.05 mmol / L). The nucleic acid dye used after the reaction was Equalbit dsDNA HS Reagent at a concentration of 0.05 mmol / L. The reaction temperature was 63℃, and the reaction time was 35 min.

[0098] The results of detecting the amplification products for different samples are as follows: Figure 10 , Figure 11As shown, 1, 2, 3, and 4 represent the results of detecting samples 1-4 together with primer set 1 and primer set 4, 5(1) represents the results of detecting sample 5 only with primer set 1 (without adding primers from primer set 4 to the reaction system), 5(4) represents the results of detecting sample 5 only with primer set 4 (without adding primers from primer set 1 to the reaction system), and 5(1+4) represents the results of detecting sample 5 together with primer set 1 and primer set 4.

[0099] according to Figure 10 , Figure 11 The results show that, among the five clinical samples tested, only sample number 5 was positive for Cryptosporidium coccidioides, meaning it contained the parasite; samples 1-4 were negative, meaning they did not contain Eimeria or Cryptosporidium coccidioides. Subsequent PCR amplification and sequencing of sample number 5 confirmed that it was indeed Eimeria. Therefore, the kit and method provided by this invention can provide technical support and assistance for the identification, epidemiological investigation, prevention and control, and purification of pathogenic Eimeria and Cryptosporidium coccidioides, as well as for the screening of vaccine raw materials.

[0100] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A CPA kit for detecting Eimeria coccidia and Cryptosporidium micrococcidia, characterized in that, Includes the primer combinations shown in SEQ ID No. 1-12.

2. The CPA kit according to claim 1, characterized in that, It also includes 2.5×pH Sensitive Reaction Buffer and Bst II DNA Polymerase; And / or, it also includes nucleic acid dyes and indicator dyes, said nucleic acid dyes including GelStain Blue or EqualbitdsDNA HS Reagent, said indicator dyes including OG Orange or Neutral Red.

3. The CPA kit according to claim 1 or 2, characterized in that, It also includes a negative control, which is a system that does not contain Eimeria coccidia nucleic acid and Cryptosporidium globosum nucleic acid; And / or, it also includes positive controls, wherein the positive controls are plasmids constructed by linking the 18S rRNA gene of Eimeria aureus and the cgd6_3910 gene of Cryptosporidium coccidioides to the pUC57 vector.

4. A primer combination for detecting Eimeria coccidia and Cryptosporidium micrococcidia, characterized in that, The primer combination includes sequences as shown in SEQ ID No. 1-12.

5. The use of the primer combination according to claim 4 in the preparation of reagents or kits for detecting Eimeria coccidia and Cryptosporidium micrococcidia.

6. A reagent containing the primer combination of claim 5.

7. A CPA method for detecting Eimeria coccidia and Cryptosporidium micrococcidia for non-disease diagnostic purposes, characterized in that, Using the DNA of the sample to be tested as a template, cross-priming isothermal amplification is performed using the CPA kit according to any one of claims 1-3, the primer combination according to claim 4, or the reagent according to claim 6.

8. The CPA method according to claim 7, characterized in that, The isothermal amplification reaction system for the cross-primers, in 25 µL volumes, comprises the following components at final concentrations: 1×pH Sensitive Reaction Buffer, 0.03–0.08 mmol / L indicator dye, 0.8–2 µmol / LF, 0.8–2 µmol / LB, 3.2–4 µmol / L CPF, 3.2–4 µmol / L CPR, 1.6–2 µmol / L QF, 1.6–2 µmol / L QR, and 600–700 units / mL Bst II DNA Polymerase; the DNA content is not less than 1 ng. Wherein, F and B are stripping primer pairs, F includes F1 and F2, the sequence of F1 is shown in SEQ ID No.1, the sequence of F2 is shown in SEQ ID No.7, and B includes B1 and B2, the sequence of B1 is shown in SEQ ID No.2, and the sequence of B2 is shown in SEQ ID No.8; CPF and CPR are cross primer pairs. CPF includes CPF1 and CPF2. The sequence of CPF1 is shown in SEQ ID No. 3, and the sequence of CPF2 is shown in SEQ ID No.

9. CPR includes CPR1 and CPR2. The sequence of CPR1 is shown in SEQ ID No. 4, and the sequence of CPR2 is shown in SEQ ID No.

10. QF and QR are detection primer pairs. QF includes QF1 and QF2. The sequence of QF1 is shown in SEQ ID No. 5, and the sequence of QF2 is shown in SEQ ID No.

11. QR includes QR1 and QR2. The sequence of QR1 is shown in SEQ ID No. 6, and the sequence of QR2 is shown in SEQ ID No.

12. And / or, after the reaction has proceeded, a step of adding nucleic acid dye may also be included.

9. The CPA method according to claim 8, characterized in that, The concentrations of F1 and F2 are the same, and the concentrations of B1 and B2 are the same. The concentrations of CPF1 and CPF2 are the same, and the concentrations of CPR1 and CPR2 are the same. The concentrations of QF1 and QF2 are the same, and the concentrations of QR1 and QR2 are the same.

10. The CPA method according to any one of claims 7-9, characterized in that, The reaction conditions for the isothermal amplification of the cross primers include: a reaction temperature of 60-65℃ and a reaction time of 15-45 min. And / or, the sample to be tested includes animal feed, cattle and sheep products, raw materials for animal vaccine production, or semi-finished animal vaccine products.

11. The CPA method according to claim 10, characterized in that, The reaction temperature is 63℃.

12. The CPA method according to any one of claims 7-9 and 11, characterized in that, If the CPA amplification product of the test sample has a clear target band in the gel electrophoresis result compared with the negative control, and / or has obvious fluorescence under ultraviolet / blue light, and / or has obvious color change reaction observed by the naked eye, then the test sample is judged to be positive; otherwise, it is judged to be negative.

13. The CPA method according to claim 10, characterized in that, If the CPA amplification product of the test sample has a clear target band in the gel electrophoresis result compared with the negative control, and / or has obvious fluorescence under ultraviolet / blue light, and / or has obvious color change reaction observed by the naked eye, then the test sample is judged to be positive; otherwise, it is judged to be negative.

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