Primer group and method for detecting cryptosporidium parvum

Abstract

The invention relates to the technical field of biology, in particular to a primer group and a method for detecting cryptosporidium parvum. The primer group for detecting the cryptosporidium parvum comprises primer nucleotide sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 6; according to the present invention, the excrement sample can be directly collected and added into the LAMP reaction reagent integrated in the microfluidic chip, the detection process is automatically completed by the full-automatic detection instrument, the spectrum is analyzed through the spectrum sensor, the collected spectrum signal is comprehensively judged through the AI technology, the full-automatic operation is achieved, the detection time can be controlled within one hour, and the detection accuracy is high. The kit realizes instant diagnosis, does not need professionals and laboratory environments, is portable and light, can be applied to instant detection in scenes such as pet hospitals on site, adopts an innovative LAMP test method, does not need nucleic acid extraction, realizes sample result output, and is low in price.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a primer set and method for detecting Cryptosporidium parvum. Background Art

[0002] Cryptosporidium parvum (CRY) is one of the pathogenic parasites causing cryptosporidiosis. To date, there are 44 recognized species and over 120 genotypes of Cryptosporidium, among which 17 species and 4 genotypes are zoonotic. Cryptosporidium parvum infects the gastrointestinal tracts of animals and humans. The main clinical manifestation of Cryptosporidium parvum infection is watery diarrhea, which usually occurs 3 - 4 days after oocyst infection and lasts for about 1 - 2 weeks. In addition, anorexia, lethargy, dehydration and even death can also be observed. Currently, there is no vaccine against Cryptosporidium; the commonly used drug for treating cryptosporidiosis is nitazoxanide, which has little effect on immunocompromised patients infected with Cryptosporidium.

[0003] The clinical symptoms and severity of cryptosporidiosis depend on the host's immune function and nutritional status. In immunocompetent individuals, infection with Cryptosporidium mainly presents as acute watery diarrhea, occasionally with mucus, generally without pus and blood, with 2 - 20 or more stools per day and a large stool volume; some people are accompanied by symptoms such as abdominal pain, abdominal distension, nausea, vomiting, thirst and fever; the course of the disease varies, ranging from 1 - 2 days to several years, with most cases lasting from 20 days to 2 months, and it is common for the disease to turn from acute to chronic and relapse. In infants and immunocompromised individuals, the symptoms after infection with Cryptosporidium are obvious and the condition is severe. Persistent cholera-like watery diarrhea is the most common, and extraintestinal organ parasitism such as the respiratory tract can also occur, and the condition is more serious and complex.

[0004] The life cycle of Cryptosporidium has 5 developmental stages: trophozoite, schizont, gametocyte, zygote and oocyst, and the oocyst is the infective stage. The oocyst is round or oval, with a diameter of 4 - 6 μm, a smooth and colorless cyst wall. The mature oocyst contains 4 crescent-shaped sporozoites and 1 residual body. In specimens stained with modified acid-fast stain, the oocysts are rose red, the background is blue-green, the sporozoites in the cyst are arranged irregularly, and the residual body is brown granular.

[0005] The life cycle of Cryptosporidium involves asexual and sexual reproduction and is completed within the same host. Oocysts are infectious upon excretion with feces. After being ingested, sporozoites are released under the action of digestive juices and invade intestinal epithelial cells to develop into trophozoites. Through multiple rounds of nuclear division, schizonts containing multiple merozoites are formed. Merozoites invade intestinal epithelial cells again and eventually develop into female and male gametocytes. After combination, zygotes are formed and develop into oocysts containing 4 sporozoites. Oocysts are divided into two types: thin-walled and thick-walled. The former can directly cause autoinfection, and the latter are excreted with feces after sporulation. The entire life cycle takes approximately 5 to 11 days.

[0006] To date, the most common and convenient method for detecting the presence of CRY is fecal ELISA (Enzyme-linked immunosorbent assay). This method is simple to operate and produces results quickly, but occasionally false positive or false negative results may occur, so further testing may be required to confirm the diagnosis. However, CRY antibodies can also be detected in healthy populations, so this method has certain limitations. Enzyme-linked immunosorbent assay can also be used to judge by detecting antigen-antibody complexes in the body, but the positive rate of this detection method is only 67%, which undoubtedly greatly limits the detection of this virus.

[0007] Veterinarians can also diagnose CRY in fecal samples through the molecular diagnostic technique - polymerase chain reaction (PCR) technique. This method is currently a relatively effective method with a relatively high detection rate of CRY. Primers targeting the conserved region of the Cryptosporidium genome are used for detection, or primers are designed based on the 18S rRNA nucleic acid sequence of Cryptosporidium parvum, and then the PCR method is used to detect Cryptosporidium parvum, and the results are specific and sensitive. Due to its high detection sensitivity, wide application range, and simple operation, the PCR technique is widely used. However, this detection technique has a complex procedure, requires sophisticated instruments, and a long detection time, which is not conducive to on-site detection in non-laboratory environments and the popularization and application in grass-roots laboratories. Summary of the Invention

[0008] In view of the above-mentioned disadvantages of the prior art, the object of the present invention is to provide a primer set and method for detecting Cryptosporidium parvum.

[0009] The present invention provides a primer set for detecting Cryptosporidium parvum on the one hand, characterized in that the nucleotide sequences of the primers included in the primer set are as follows:

[0010] SEQ ID NO.1: 5’-CTTTGGGTTCTGGGGGGA-3’;

[0011] SEQ ID NO.2: 5’-GGTCTCGTTCGTTAACGGAATT-3’;

[0012] SEQ ID NO.3:

[0013] 5’-TCAAATTAAGCCGCAGGCTCCACAAAATGGTCGCAAGGCTGAAAC-3’;

[0014] SEQ ID NO.4:

[0015] 5’-ACGGGAAAACTCACCAGGTCCAAAAACATGCACCACCACCCATAG-3’;

[0016] SEQ ID NO.5: 5’-GGTGCCCTTCCGTCAATTC-3’;

[0017] SEQ ID NO.6: 5’-GGAAGGATTGACAGATTGATAGCTC-3’.

[0018] Among them, the forward outer primer F3 has the nucleotide sequence shown in SEQ ID NO.1; the reverse outer primer B3 has the nucleotide sequence shown in SEQ ID NO.2; the forward inner primer FIP has the nucleotide sequence shown in SEQ ID NO.3; the reverse inner primer BIP has the nucleotide sequence shown in SEQ ID NO.4, the loop primer LF has the nucleotide sequence shown in SEQ ID NO.5, and the loop primer LB has the nucleotide sequence shown in SEQ ID NO.6.

[0019] Specifically, the present invention also provides the application of the above primer set. The above primer set can be applied to the preparation of a Cryptosporidium parvum kit, or the application of detecting Cryptosporidium parvum using the primer set, and the application of the above primer set in a microfluidic chip for detecting Cryptosporidium parvum, especially the detection of Cryptosporidium parvum using a loop-mediated isothermal amplification - spectral sensor - artificial intelligence technology platform.

[0020] On the other hand, the present invention also provides a method for detecting Cryptosporidium parvum, including: performing loop-mediated isothermal amplification on a sample to be tested using LAMP detection technology in combination with the above primer set; analyzing the amplification product.

[0021] Further, the reaction temperature of the loop-mediated isothermal amplification is 65°C, and the reaction time is 35 minutes.

[0022] Further, the reaction of the loop-mediated isothermal amplification is carried out in a microfluidic chip.

[0023] Further, the method for analyzing the amplification product includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction result.

[0024] The beneficial effect of the present invention is that the present invention innovatively develops a set of specific primer formulas for LAMP reaction that do not require nucleic acid extraction and can directly use fecal samples for Cryptosporidium parvum 18S rRNA gene sequences, and collects data through spectral sensors to perform rapid nucleic acid detection methods for Cryptosporidium parvum. The method can directly use pet fecal samples and add them to the LAMP reaction reagent integrated in the microfluidic chip. The detection process is automatically completed by a fully automatic detection instrument, and the spectrum is analyzed by a spectral sensor. The collected spectral signals are comprehensively judged by AI technology to achieve fully automatic operation, control the detection time within one hour, and achieve instant diagnosis. It does not require professionals and laboratory environments, is portable and lightweight, and can be used on-site for immediate detection in pet hospitals and other scenarios. It adopts an innovative LAMP test method, does not require nucleic acid extraction, and achieves sample-in and result-out, and is low-priced.

[0025] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention are realized and obtained by the structures particularly pointed out in the description and the drawings.

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is the screening result of LAMP reaction of 5 primer sets designed for the 18S rRNA gene sequence of Cryptosporidium parvum;

[0029] Figure 2 It is the test result of the present invention for CRY positive samples and negative samples;

[0030] Figure 3 is the test result of the specific experiment of the present invention;

[0031] Figure 4 It is the detection result of the Cryptosporidium parvum detection method for fecal samples of the present invention. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0033] In the embodiments of the present invention, the CRY standard positive sample is a synthesized CRY-18S rRNA gene sequence positive plasmid, Salmonella (Sal), Toxoplasma gondii (TOX), and Canine parvovirus (CPV) are all synthesized plasmid samples, and the primer sequences involved are all ordered from Sangon Biotech (Shanghai) Co., Ltd.

[0034] 1. Design and screening of primers

[0035] By using a relatively high concentration of the CRY standard positive sample, the following LAMP primer sets designed with the CRY-18S rRNA nucleic acid fragment as the template were screened. The goal was to screen out a set of primers with the best sensitivity and specificity.

[0036] The primer design method is as follows:

[0037] (1) Download the Cryptosporidium parvum 18S rRNA gene sequence (Sequence ID: AF112571.1) from NCBI as the primer design template.

[0038] The 18S rRNA nucleic acid sequence (SEQ ID NO.7) is:

[0039]

[0040] (2) Use the software PrimerExplorer V5

[0041] (http: / / primerexplorer.jp / lampv5e / index.html) to design specific primers using the Cryptosporidium parvum - 18S rRNA gene sequence as a template. Based on six different regions on the Cryptosporidium parvum - 18S rRNA gene sequence labeled as F3, F2, F1, B1, B2, B3 in sequence from the 5' end, design LAMP reaction primers. Among them, F1, F2, F3, B1, B2, B3 are sequence fragments approximately 20 bp long on the target gene, and F1c and B1c are the complementary regions of F1 and B1 respectively. The four primers include: forward outer primer F3 (Forward Outer Primer), backward outer primer B3 (Backward Outer Primer), forward inner primer FIP (Forward Inner Primer), and backward inner primer BIP (Backward Inner Primer). At the same time, use the free software PrimerExplorer V5 to design 1 - 2 loop primers LF (Loop Primer Forward) and LB (Loop Primer Backward) between F1c and F2c, or between B1c and B2c to accelerate the LAMP reaction rate. The primers are required to meet the conditions that the Tm values (DNA strand melting temperature, melting temperature) of F1c and B1c are 64°C - 66°C, the Tm values of F2, F3, B2, B3 are 59°C - 61°C, and the Tm values of the loop primers are 64°C - 66°C. The free energies at the 3' ends of F2 / B2, F3 / B3 and LF / LB and the 5' ends of F1c and B1c should not be greater than -4 kcal / mol. The free energy change (ΔG) is equal to the free energy of the product after the primer binds to the template minus the free energy of the initial reactants. The smaller the ΔG (the larger the absolute value of the negative number), the easier it is for the primer to bind to the template. And the GC content in the primer sequence should be controlled at 45% - 65%. At the same time, ensure that each primer, especially the inner primer, is not prone to forming a secondary structure to prevent the formation of primer dimers. There should be an appropriate spacing between the sequence fragments recognized by each primer. The spacing between the 3' end of F2 and the 3' end of B2 (LAMP amplification region) is 120 - 160 bases, the spacing between the 5' end of F2 and the 5' end of F1 (the region forming the loop structure) is 40 - 60 bases, and the spacing between F2 and F3 is 0 - 60 bases. All the above conditions can be automatically calculated by the free software PrimerExplorer V5 and a list of the designed primers is given;

[0042] (3) Select multiple primer sets designed by the software PrimerExplorer V5 with different positions on the Cryptosporidium parvum - 18S rRNA gene sequence as templates. By inputting the target product sequences of different primer sets into the NCBI NucleotideBLAST

[0043] (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) online tool, analyze the specificity of the LAMP products corresponding to the primer sets to ensure that there is no non-specific amplification in the pet genome and other pathogens.

[0044] Through the above method, the sequences of 5 groups of LAMP primer sets were initially designed for selection. The nucleotide sequences of each group of primers are shown in Table 1 below:

[0045] Table 1 Primer Design

[0046]

[0047]

[0048] Using the above 6 groups of primer sets, with the CRY standard positive sample as the positive sample and sterilized deionized water (ddH2O) as the negative sample, conduct LAMP amplification experiments, and observe the color changes of the LAMP reaction system of each primer set. As Figure 1 shown, the primer set of the second group changed color (from purple-red to bright yellow) in a shorter time for the positive sample (+), and the negative sample (-) did not change color at 35 min, indicating that this primer set has high sensitivity and specificity. For other primer sets, chaotic positive and negative reactions occurred, so they were not selected. In summary, the second group is the primer set with the best sensitivity and specificity. Therefore, the second group of primer sets was selected as the primer set for detecting Cryptosporidium parvum.

[0049] 2. Reaction Conditions

[0050] After several rounds of testing different reaction conditions, the following conditions were finally selected as the final reagent ratio and reaction conditions:

[0051] (1) Preparation of 10× primer mixture:

[0052] Table 2 Primer set mixed concentration ratio

[0053] Working concentration (μM) 10× Primer Mix Concentration (μM) FIP 1.6 16 BIP 1.6 16 F3 0.2 2 B3 0.2 2 LB 0.4 4 LF 0.4 4

[0054] Table 3

[0055] Primer Concentration (μM) Primer Name For 40 Reactions (μL) 100 FIP 8 100 BIP 8 10 F3 10 10 B3 10 100 LB 2 100 LF 2 Sterilized Deionized Water 10 Total Volume (μL) 50

[0056] (2) Primer, 2×LAMP premix and positive / negative sample ratio in the LAMP reaction system:

[0057] Table 4

[0058] For 1 Reaction (μL) 2× LAMP Premix 6.25 10× Primer Mix 1.25 Positive / Negative Sample 5.0

[0059] The 2×LAMP premix is a well-known formula, containing MgSO4 (the concentration of MgSO4 is 6 mM - 12 mM), buffer, dNTPs, KCl, (NH4)2SO4, Bst DNA polymerase, phenol red, sterile enzyme-free water, etc.

[0060] The positive sample can be a CRY standard positive sample, that is, the synthesized CRY-18S rRNA gene sequence positive plasmid.

[0061] The negative sample can be sterile deionized water (ddH2O).

[0062] (4) Reaction conditions

[0063] Under the temperature condition of 65 °C, constant temperature amplification for 35 minutes gives better detection results.

[0064] Heating can be carried out using a constant temperature heating device (water bath, metal bath, constant temperature oven, or PCR instrument, etc.).

[0065] 3. LAMP detection method for Cryptosporidium parvum (CRY)

[0066] The present invention uses a CRY standard positive sample (the synthesized CRY-18S rRNA gene sequence positive plasmid) and performs detection according to the following steps:

[0067] The first step: Preparation of CRY positive / negative samples

[0068] Dilute the synthesized CRY-18S rRNA gene sequence positive plasmid with sterile deionized water, and dilute the concentration of the CRY standard positive sample to 1000 copies / μL as the positive sample; use sterile deionized water (ddH2O) as the negative sample.

[0069] The second step: Prepare a 10× primer mixture according to the method in Table 3 above.

[0070] The third step: Take the Cryptosporidium parvum positive / negative samples prepared in the first step and prepare the LAMP reaction systems for each reaction according to the method in Table 4 above.

[0071] The fourth step: Heat at 65 °C for 35 minutes.

[0072] The reaction results are as Figure 2 shown. Among them, the LAMP reaction systems of 20 negative samples (below) are all purplish red, and the LAMP reaction systems of 20 positive samples (above) all turn yellow. The positive accuracy rate in the reaction results is 100%, and the negative accuracy rate is 100%. It can be seen that Cryptosporidium parvum can be clearly detected.

[0073] 4. Specificity experiment

[0074] To detect the specificity of the primer set for detecting Cryptosporidium parvum, plasmid solutions of Salmonella (Sal), Toxoplasma gondii (TOX), and Canine parvovirus (CPV) with a concentration of 1000 copies / μL were used as cross-detection samples respectively.

[0075] The first step: Preparation of CRY positive / negative samples for cross-testing

[0076] Positive sample: The synthesized CRY-18S rRNA gene sequence positive plasmid with a dilution concentration of 1000 copies / μL was used as the positive sample;

[0077] Negative sample: Plasmid solutions of Salmonella (Sal), Toxoplasma gondii (TOX), and Canine parvovirus (CPV) with a dilution concentration of 1000 copies / μL were used as negative samples respectively.

[0078] The second step: Prepare the 10× primer mixture according to the method in Table 3 above.

[0079] The third step: Respectively take the positive sample and each negative sample prepared in the first step, and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0080] The fourth step: Heat at 65°C for 35 minutes.

[0081] The reaction results are as Figure 3 shown. The LAMP reaction systems of 4 positive samples containing the synthesized CRY-18S rRNA gene sequence positive plasmid solution all turn yellow. 4 negative samples containing Salmonella (Sal) plasmid solution, 4 negative samples containing Toxoplasma gondii (TOX) plasmid solution, and 4 negative samples containing Canine parvovirus (CPV) plasmid solution do not change color. It can be seen that no false positive results appear, proving that the primer set for detecting Cryptosporidium parvum of the present invention has good specificity.

[0082] According to Figure 2 and Figure 3 of the experimental results, the sensitivity and specificity of LAMP for detecting Cryptosporidium parvum are preliminarily calculated. According to Figure 2 and Figure 3Statistical analysis of the experimental results showed that all 24 positive samples out of 24 presented clear positive results. Therefore, the sensitivity was calculated as: Among the 32 negative samples of 12 other bacterial species and 20 sterilized deionized water (ddH2O), all presented clear negative results. Therefore, the specificity was calculated as:

[0083] 5. Loop-mediated isothermal amplification (LAMP) assay for Cryptosporidium parvum in fecal samples

[0084] Step 1: Preparation of positive / negative fecal samples

[0085] Insert a cotton swab into different positions of the fecal sample 5 - 8 times. When inserting, fully immerse the head of the cotton swab into the fecal sample and try to cover all parts of the fecal sample. If there is a large amount of solid feces on the cotton swab, gently scrape off the excess fecal solid; completely immerse the cotton swab dipped in the fecal sample into 1 mL of pre-prepared sterilized deionized water, and shake the cotton swab dozens of times to dissolve the fecal sample in the water to obtain a negative fecal sample.

[0086] Add a CRY standard positive sample (synthetic CRY-18S rRNA gene sequence plasmid) with a certain dilution ratio to a part of the above-prepared negative fecal samples to obtain positive fecal samples.

[0087] Step 2: Prepare the 10× primer mixture according to the method in Table 3 above.

[0088] Step 3: Dilute the positive / negative fecal samples prepared in Step 1 with sterilized deionized water at a ratio of 1 / 50, and make the concentration of the synthetic CRY-18S rRNA gene sequence plasmid in the positive fecal samples 1000 copies / μL; prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0089] Step 4: Heat at 65°C for 35 minutes.

[0090] The reaction results are as Figure 4 shown. All 10 negative fecal samples (-) presented clear negative results, and all 10 positive fecal samples (+) turned yellow. It can be seen that Cryptosporidium parvum in fecal samples can be clearly detected.

[0091] For the LAMP assay for Cryptosporidium parvum in fecal samples, the method of collecting and processing fecal samples is simple, without nucleic acid extraction, which simplifies the detection steps and shortens the detection time. It only takes about 1 hour from sample collection to obtaining the detection results.

[0092] This detection method can also detect Cryptosporidium parvum through the Loop-mediated Isothermal Amplification-Spectral Sensor-Artificial Intelligence (LAMP-SpectralSensor-AI) technology platform. The diluted sample to be tested can be directly injected into the LAMP reaction reagent integrated in the reaction chamber of the microfluidic chip for amplification. The amplification product is analyzed by a spectral sensor, and the collected spectral signals are comprehensively judged by AI technology to quickly determine whether there is Cryptosporidium parvum in the sample. Moreover, the spectral detection method combined with the AI algorithm can achieve a detection accuracy at the molecular detection level. By confining the sample to be tested in the microfluidic environment of the device, the risk of sample contamination is reduced, and the sample volume and reagents required for detection are minimized, thus further reducing the overall cost of screening and detection. This technology requires no complex operations and can be completed by the medical staff in pet hospitals through simple training. It can achieve fully automated detection from sample input to result output and is suitable for rapid point-of-care testing in scenarios such as pet hospitals.

[0093] Inspired by the ideal embodiments of the present invention described above, through the above description, relevant staff can make various changes and modifications within the scope not deviating from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A primer set for detecting Cryptosporidium parvum, characterized in that: The nucleotide sequences of the primers included in the primer set are as follows: SEQ ID NO.1: 5'-CTTTGGGTTCTGGGGGGA-3'; SEQ ID NO.2: 5'-GGTCTCGTTCGTTAACGGAATT-3'; SEQ ID NO.3: 5'-TCAAATTAAGCCGCAGGCTCCACAAAATGGTCGCAAGGCTGAAAC-3'; SEQ ID NO.4: 5'-ACGGGAAAACTCACCAGGTCCAAAAACATGCACCACCACCCATAG-3'; SEQ ID NO.5: 5'-GGTGCCCTTCCGTCAATTC-3'; SEQ ID NO. 6: 5'-GGAAGGATTGACAGATTGATAGCTC-3'.

2. Use of the primer set according to claim 1 in preparing a Cryptosporidium parvum detection kit.

3. Use of the primer set according to claim 1 in detecting Cryptosporidium parvum.

4. Use of the primer set according to claim 1 in a microfluidic chip for detecting Cryptosporidium parvum.

5. A method for detecting Cryptosporidium parvum, characterized in that: include: Using LAMP detection technology combined with the primer set as claimed in claim 1 to perform loop-mediated isothermal amplification on the sample to be tested; Analyze the results of the amplification products.

6. The method for detecting Cryptosporidium parvum according to claim 5, characterized in that: The reaction temperature of the loop-mediated isothermal amplification was 65° C., and the reaction time was 35 minutes.

7. The method for detecting Cryptosporidium parvum according to claim 5, characterized in that: The loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

8. The method for detecting Cryptosporidium parvum according to claim 5, characterized in that: The method for analyzing the results of the amplification products includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction results.

Images