Primer sets and probes for triple real-time fluorescence quantitative PCR detection of Lawsonia intracellularis, Salmonella, and Isospora suis, and detection kits

Abstract

The invention belongs to the biological field, and discloses a primer set and a probe for triple real-time fluorescence quantitative PCR detection of intracellular Lawsonia, Salmonella and Isospora suis. The sequences of the primers and probes for specific amplification of intracellular Lawsonia are shown in SEQ ID NO.1 to SEQ ID NO.3; the sequences of the primers and probes for specific amplification of Salmonella are shown in SEQ ID NO.4 to SEQ ID NO.6; the sequences of the primers and probes for specific amplification of Isospora suis are shown in SEQ ID NO.7 to SEQ ID NO.9. The primer set, the probe and the reaction system can be used to detect intracellular Lawsonia, Salmonella and Isospora suis with high sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and more specifically, relates to a primer set and a probe and a detection kit for triple real-time fluorescence quantitative PCR detection of Lawsonia intracellularis, Salmonella and Isospora suis. Background Art

[0002] Diarrhea is one of the most common syndromes in pig production, with huge economic losses and complex etiology. Lawsonia intracellulari (LI) is an intracellular obligate anaerobe that causes pig proliferative enteropathy (PPE), characterized by immature enterocyte adenoid hyperplasia in the crypts of the ileum and colon. Porcine proliferative enteritis, also known as porcine ileitis, can be divided into three types according to clinical manifestations and pathological changes. One is proliferative hemorrhagic enteropathy (PHE), characterized by acute hemorrhagic diarrhea, and the pathological characteristics are hyperplastic hypertrophy and hemorrhage of the ileal and colonic mucosa; porcine adenomatosis (PIA) causes anorexia, diarrhea and poor growth in young pigs; the other is necrotizing enteritis (NE), characterized by severe mucosal thickening and brown-yellow necrotic foci on the surface of the pig's intestine. PHE is an acute form of PPE, mainly infecting fattening pigs and breeding pigs aged 4-12 months; PIA and NE are chronic forms of PPE, usually infecting pigs aged 6-20 weeks. The overall clinical manifestations of sick pigs are decreased appetite, slowed growth rate, poor uniformity, diarrhea, etc. Functionally, it manifests as nutritional absorption disorders and decreased production levels. Clinically, it is often accompanied by mixed infections with other pathogens, such as Brachyspira hyodysenteriae, Brachyspira coli, Salmonella, pathogenic Escherichia coli, etc.

[0003] Swine paratyphoid, also known as swine salmonellosis, is an infectious disease of piglets caused by Salmonella enterica. The main pathogens are Salmonella choleraesuis and Salmonella typhisuis. Salmonella typhimurium, Salmonella derby and Salmonella enteritidis are also common causes. The disease is contagious, harmful, spreads quickly and has a high mortality rate. Pigs of all ages can be infected, and it is common in piglets under 6 months old. The incidence and mortality rate of pigs aged 2-4 months are high. Sick pigs become stiff pigs after surviving the disease, and their growth and development are slow.

[0004] Coccidiosis in pigs is a highly contagious group parasitic disease. The main pathogens are Eimeria tijuni, Eimeria crassa, Eimeria spinosa and Isospora suis (IS), among which Isospora suis has the strongest pathogenicity. Isospora suis can infect pigs at different growth stages, among which suckling piglets are the most susceptible, followed by middle-aged pigs, and most adult pigs are carriers. Coccidia diarrhea causes the intestinal villi to shorten or even fall off, and at the same time causes other intestinal pathogens (such as Escherichia coli, Salmonella, Haemophilus parasuis, epidemic diarrhea, infectious gastroenteritis, etc.) to aggravate piglet diarrhea, and in severe cases lead to death. On the other hand, it seriously affects the weaning of piglets, and then affects the age of market release, and affects the production performance of pigs during the growth and fattening period.

[0005] In the prior art, there is no relevant literature recording a scheme for simultaneously detecting Lawsonia intracellularis, Salmonella, and Isospora suis.

[0006] In the prior art, the following solutions are disclosed:

[0007] CN115058528A discloses a composition, a kit, a method and its use for detecting and typing swine diarrhea pathogens, wherein the composition detects Salmonella choleraesuis (SC), Lawsonia intracellularis (LI), Brachyspira hyodysenteriae (BH), pathogenic Escherichia coli (ETEC), Clostridium perfringens (Cp) and Clostridium difficile (Cd) through multiplex fluorescence PCR.

[0008] The above protocol does not record the detection of Isospora suis.

[0009] Intracellular Lawsonia, Salmonella, and Isospora suis are prevalent worldwide, causing serious losses to the aquaculture economy, and have a strong demand for detection. Compared with the high incidence of viral diarrhea (such as porcine epidemic diarrhea, porcine transmissible gastroenteritis, porcine Deta coronavirus, porcine rotavirus, etc.), diarrhea caused by bacteria and parasites is often ignored. Especially porcine ileitis and Isospora suis, the incidence rate of large-scale pig farms in my country has increased year by year, and it has become a spreading trend; and salmonellosis is a zoonosis. Due to the improper use of antibiotics, the drug resistance of Salmonella continues to rise, and multi-drug resistant strains continue to appear, seriously threatening public health safety. From the age of onset, the growth stages of these three pathogens infecting pigs are similar, and mixed infection is prone to occur. Therefore, based on the actual situation of the above production and prevention and control, the triple qPCR detection method of intracellular Lawsonia, Salmonella and Isospora suis established by the present invention can quickly detect and distinguish the above three pathogens, providing a reference for clinical prevention and control. Summary of the invention

[0010] The main purpose of the present invention is to provide a primer set and probe for triple real-time fluorescence quantitative PCR detection of intracellular Lawsonia solium, Salmonella and Isospora suis, and a detection kit. The primer set, probe and reaction system can be used to highly sensitively and specifically detect intracellular Lawsonia solium, Salmonella and Isospora suis.

[0011] According to a first aspect of the present invention, the present invention provides a primer set and probe for triple real-time fluorescence quantitative PCR detection of Lawsonia intracellularis, Salmonella, and Isospora suis, and the sequences of the primers and probes for specifically amplifying Lawsonia intracellularis are shown in SEQ ID NO.1 to SEQ ID NO.3;

[0012] The sequences of primers and probes for specific amplification of Salmonella are shown in SEQ ID NO.4 to SEQ ID NO.6;

[0013] The sequences of primers and probes for specific amplification of Isospora suis are shown in SEQ ID NO.7 to SEQ ID NO.9.

[0014] The detection target of intracellular Lawsoniae of the present invention is the dnaA gene site; for intracellular Lawsoniae, common detection targets in the art are aspA (aspartate ammonia-lyase), ubiE, and 16S rDNA; there are few reports in the literature on the dnaA gene site as a specific detection target;

[0015] The serotypes of swine-derived Salmonella are very complex. According to the different characteristics of O antigen and H antigen, Salmonella serotypes can be roughly divided into 2631 serotypes. The most popular serotypes of clinical diseases caused by Salmonella in pigs are Salmonella choleraesuis, Salmonella typhisuis, and Salmonella typhimurium. However, since the complete gene sequence of Salmonella typhisuis has not been published on NBCI, the detection sites selected by the present invention are brand-new detection primers and sites developed for Salmonella.

[0016] Coccidiosis in pigs is a highly contagious group parasitic disease. The main pathogens are Eimeria tiliaceus, Eimeria crassa, Eimeria spinosa and Isospora suis, among which Isospora suis has the strongest pathogenicity. Since there are no characteristic symptoms in the early stage of Isospora suis infection, and there is no obvious regularity in the amount of oocysts discharged after infection and the characteristics of short oocyst discharge cycle, rapid detection is still the bottleneck for effective diagnosis of the disease. However, there is no report on the complete genome of Isospora suis so far, and even its chromosome number is not yet known. For the study of the genomics of Isospora coccidia, there are only a few reports on its ribosome and mitochondrial genome sequences. At present, the most commonly used PCR method for detecting Isospora suis is to design specific primers based on the 18S rRNA gene sequence of Isospora suis recorded in the NCBI database, thereby establishing a PCR detection method. The present invention designs targeted primers and probes for the ITS2 gene (433bp) of the ribosomal RNA gene sequence of Isospora suis.

[0017] Regarding this site, the prior application CN107043809B of our Academy of Agricultural Sciences disclosed a method for constructing a quantitative detection of Isospora suis by qRT-PCR, in which the detection site is the IS-ITS2 gene sequence, which can detect a single worm egg; however, after the primers and probes shown in the invention were applied to the present invention, we found that there was a relatively obvious antagonism.

[0018] At the same time, the present invention also discloses a triple real-time fluorescence quantitative PCR detection kit for detecting intracellular Lawsonia intracellularis, Salmonella, and Isospora suis, wherein the reaction system comprises the primer set and probe as described above and 2×AceQUniversalU + Probe Master Mix V2, ddH 2 O.

[0019] In the above triple real-time fluorescence quantitative PCR detection kit, the reaction system contains 2×AceQUniversal U +Probe Master Mix V2 total 1 0μL;

[0020] 1 μL of each of the upstream primer and the downstream primer of the primers with a concentration of 10 μM for specific amplification of Lawsonia intracellularis;

[0021] 0.2 μL of the probe that specifically amplifies Lawsonia intracellularis at a concentration of 10 μM;

[0022] 0.8 μL of each of the upstream primer and the downstream primer of the primers with a concentration of 10 μM for specific expansion of Salmonella;

[0023] The probe for specific amplification of Salmonella at a concentration of 10 μM was 0.3 μL;

[0024] The concentration is 10 μM, and the upstream primer and the downstream primer of the primers for specific amplification of Isospora suis are 0.2 μL each;

[0025] The probe that specifically amplifies Isospora suis at a concentration of 10 μM was 0.3 μL.

[0026] In the above triple real-time fluorescence quantitative PCR detection kit, the reaction conditions applicable to the reaction system are: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 55°C for 30 seconds, and 45 cycles.

[0027] One of the above technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0028] The present invention takes Lawsonia intracellularis, Salmonella and Isospora suis which cause swine ileitis, swine paratyphoid and swine coccidiosis as research objects, uses bioinformatics and biological techniques to screen primer sets and probes respectively and optimize the concentrations of primers and probes, and successfully establishes a triple real-time fluorescence quantitative PCR detection method.

[0029] The method provided by the present invention saves detection costs and time. The detection of three pathogens is completed in one reaction system, which greatly avoids aerosol pollution.

[0030] At the same time, the method provided by the present invention has good sensitivity and specificity, and has good repeatability and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0032] Figure 1 This is the standard curve of triple real-time fluorescence quantitative PCR;

[0033] Figure 2 The sensitivity test results of primers and probes for Lawsonia intracellularis;

[0034] Figure 3 The sensitivity test results of primers and probes for Salmonella;

[0035] Figure 4 The results of the sensitivity test of primers and probes for Isospora suis;

[0036] Figure 5 The fluorescence curves amplified by the primers and probes for Lawsonia intracellularis;

[0037] Figure 6 The fluorescence curve amplified by the primers and probe for Salmonella;

[0038] Figure 7 This is the fluorescence curve amplified by the primers and probe of Isospora suis;

[0039] Figure 8 It is the melting curve of three pairs of primers in SYBR Green QPCR system simultaneously;

[0040] Fig. 9 is the melting curve of the first pair of primers in the specificity screening process of the primers for Lawsonia intracellularis in Comparative Example 1;

[0041] Fig.10 is the melting curve of the second pair of primers in the specificity screening process of the primers for Lawsonia intracellularis in Comparative Example 1;

[0042] Fig.11 is the melting curve of the third pair of primers in the specificity screening process of the primers for Lawsonia intracellularis in Comparative Example 1;

[0043] Fig.12 is the melting curve of the first pair of primers in the specific screening process of Salmonella primers in Comparative Example 2;

[0044] Fig.13 is the melting curve of the second pair of primers in the specific screening process of Salmonella primers in Comparative Example 2;

[0045] Fig.14 is the melting curve of the third pair of primers in the specific screening process of Salmonella primers in Comparative Example 2;

[0046] Fig.15 is the melting curve of the first pair of primers in the specificity screening process of primers for Isospora suis in Comparative Example 3;

[0047] Fig.16 is the melting curve of the second pair of primers in the specificity screening process of primers for Isospora suis in Comparative Example 3;

[0048] Fig.17is the melting curve of the third pair of primers in the specificity screening process of primers for Isospora suis in Comparative Example 3;

[0049] Fig.18 The results of verifying whether the three pairs of primers in Comparative Example 4 will produce dimers in SYBR Green QPCR;

[0050] Fig.19 This is the single-plex TaqMan QPCR amplification curve of Lawsonia intracellularis in Comparative Example 5;

[0051] Fig. 20 This is the single-plex TaqMan QPCR amplification curve of Salmonella in Comparative Example 5;

[0052] Fig.21 This is the single-plex TaqMan QPCR amplification curve of Isospora suis in Comparative Example 5;

[0053] Fig. 22 is the amplification curve of intracellular Lawsoniae in the dual system of intracellular Lawsoniae / Isospora suis of Comparative Example 5;

[0054] Fig.23 is the amplification curve of Isospora suis in the dual system of Lawsonia intracellularis / Isospora suis of Comparative Example 5;

[0055] Fig.24 This is the amplification curve of Salmonella in the Salmonella / Isospora suis dual system of Comparative Example 5;

[0056] Fig.25 This is the amplification curve of Isospora suis in the Salmonella / Isospora suis dual system of Comparative Example 5;

[0057] Fig.26 This is the plasmid map of recombinant LI-SE-IS. DETAILED DESCRIPTION

[0058] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout.

[0059] The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, but should not be construed as limiting the present invention.

[0060] The materials used in the following examples and test examples include:

[0061] The primer set and probe for intracellular Lawsonia solium, Salmonella typhimurium, and Isospora suis of the present invention, and the recombinant LI-SE-IS plasmid (the plasmid map of which is referenced to Fig.26 ) were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.;

[0062] 2×AceQ Universal U + Probe Master Mix V2 was purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0063] Fecal genomic DNA / RNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.;

[0064] Tianlong Fluorescence Quantitative PCR Instrument was purchased from Xi'an Tianlong Technology Co., Ltd.

[0065] LC480 Roche real-time fluorescence quantitative PCR instrument was purchased from ROCHE.

[0066] Example 1

[0067] The detection method includes the following steps

[0068] The genome sequences of Lawsonia intracellularis, Salmonella, and Isospora suis were analyzed by bioinformatics, and primers and probes were designed for the conserved nucleotide sequence dnaA fragment of Lawsonia intracellularis, the conserved nucleotide sequence InvA fragment of Salmonella, and the conserved nucleotide sequence ITS2 fragment of Isospora suis using primerpremier 5 software, as shown in Table 1.

[0069] Table 1. Triple real-time PCR primers and probes

[0070]

[0071] preparing a reaction system;

[0072] The reaction system includes the recombinant LI-SE-IS plasmid as a template, 2×AceQ Universal U + ProbeMaster Mix V2, LI-2F, LI-2R, LI-P, SE-1F, SE-1R, SE-P, IS-2F, IS-2R, IS-2P, ddH 2 O, 20 μL in total; 2×AceQ Universal U + Probe Master Mix V2 10μL; LI-F and LI-R (10μM) 1μL each; LI-P (10μM) 0.2μL; SE-F and SE-R (10μM) 0.8μL each; SE-P (10μM) 0.3μL; IS-F and IS-R (10μM) 0.2μL each; IS-P (10μM) 0.3μL; ddH 2 O; set to ddH 2 O is the template as a negative control. The total system is 20 μL.

[0073] The reaction system was used for real-time fluorescence quantitative PCR according to the following reaction conditions: 95°C pre-denaturation for 5 min, 95°C denaturation for 10 sec, 55°C annealing and extension for 30 sec for 45 cycles, and the fluorescence signals of the three channels of FAM, VIC, and CY5 were collected simultaneously in the last step of each cycle.

[0074] Example 2

[0075] Optimization of triple real-time fluorescence quantitative PCR reaction conditions

[0076] Using the recombinant LI-SE-IS plasmid as a template, the matrix method was used to optimize the primers, probe concentrations, annealing temperature and other conditions of real-time fluorescence quantitative PCR on a Tianlong fluorescence quantitative PCR instrument (Xi'an Tianlong Technology Co., Ltd.).

[0077] It is difficult to avoid the generation of primer dimers when three pairs of primers are in the same system. Figure 8 , Figure 8 The figure is the melting curve of three pairs of primers in the SYBR Green QPCR system at the same time. The lower peak at 75-80℃ is due to nonspecific amplification caused by primer dimers, and the peak at 80-84℃ is the main amplification peak. In order to reduce the impact caused by it, the concentration (ratio) of three pairs of primers and probes was optimized using the matrix method in the TaqMan QPCR method. First, the primers and probes of Lawsonia intracellularis, Salmonella, and Isospora suis were optimized respectively. The best two sets of primers and probes were selected according to the CT value. Then, the best primers and probes of the three pathogens were cross-combined (a total of 8 groups) for reaction, and the combination with the least impact on the CT value, fluorescence signal intensity, and amplification curve of the three pathogens was selected.

[0078] Using the recombinant LI-SE-IS plasmid as a template, the primer was explored between 0.1-0.5μM, i.e., 0.2-1μL (10μM) / reaction. The probe concentration was 0.1-0.25μM, i.e., 0.2-0.5μL (10μM) / reaction. The reaction conditions for real-time fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min, 95℃ denaturation for 10sec, 55℃ annealing extension for 30sec, and a total of 45 cycles. The fluorescence signals of the three channels of FAM, VIC, and CY5 were collected simultaneously in the last step of each cycle. After the reaction, the fluorescence curve and CT value were obtained. The optimal triple real-time fluorescence quantitative PCR reaction conditions were selected based on the amplification curve with a lower CT value and a higher fluorescence value. The optimal triple real-time fluorescence quantitative PCR reaction system screened out through experimental comparison and system optimization is shown in Table 2.

[0079] Table 2. Optimal triple real-time fluorescence quantitative PCR reaction system

[0080]

[0081] Establishment of triple real-time fluorescence quantitative PCR standard curve: Using the copy number formula (6.02×10 23 )×(ng / μL×10 -9 / (DNA length×660)=copies / μL to calculate the copy number of the recombinant LI-SE-IS plasmid. Then, the recombinant LI-SE-IS plasmid was serially diluted 10 times to a plasmid concentration of 1×10 7 -1×10 2 copies / μL, set 3 replicates for each concentration, and perform the test according to the real-time quantitative PCR operation procedure to obtain a standard curve. Figure 1 As shown, the standard curve equations of Lawsonia intracellularis are Y = -3.587*X + 41.11, R 2 =0.9997, E = 90.013%; the standard curve equations for Salmonella are Y = -3.604*X + 42.09, R 2 =0.9997, E = 89.439%; the standard curve equations of Isospora suis are Y = -3.659*X + 40.92, R 2 =0.9992, E=87.628%, indicating that there is a high degree of credibility and a good linear relationship between the plasmid copy number and the CT value.

[0082] Example 3

[0083] Triple real-time fluorescence quantitative PCR sensitivity test

[0084] The recombinant LI-SE-IS plasmid was diluted 10-fold in series to a plasmid concentration of 1×10 7 -1×10 0 copies / μL, set 3 replicates for each concentration, and perform the test according to the triple real-time quantitative PCR operation procedure to determine the minimum detection amount and set up a negative control. Figure 2 , 3 As shown in Figure 4, based on the test results, the sensitivity information of the three sets of primers and probes can be obtained: the minimum detection threshold of intracellular Lawsonia spp. is 1×10 1 copies / μL, and the minimum detection threshold for Salmonella is 1×10 1 copies / μL, and the minimum detection threshold for Isospora suis is 1×10 1 copies / μL.

[0085] Example 4

[0086] Repeatability test of triple real-time fluorescence quantitative PCR

[0087] Use recombinant LI-SE-IS plasmid 1×10 3 copies / μL, 1×10 4 copies / μL, 1×10 5 copies / μL, and 3 concentration gradients were used as templates for repeatability test. Intra-batch repeatability test: The plasmid of each concentration gradient was repeated 3 times using the triple real-time fluorescence quantitative PCR method of the present invention, and a negative control was set up; Intra-batch repeatability test: The samples of each concentration gradient were tested under the same conditions in 3 different time periods using the triple real-time fluorescence quantitative PCR method of the present invention. At the same time, the intra-group and inter-group coefficient of variation (CV) was calculated based on the CT value of each concentration gradient, and the coefficient of variation (CV) = standard deviation (S) / mean The repeatability of the triple real-time fluorescence quantitative PCR of the present invention was evaluated in this way. As shown in the figure, the coefficient of variation of the intra-batch repeat test was between 0.053% and 0.429%, and the coefficient of variation of the inter-batch repeat test was between 0.646% and 1.167%, which had relatively good repeatability and stability.

[0088] Table 3. Intra-batch reproducibility test of triple real-time fluorescence quantitative PCR

[0089]

[0090] Table 4. Triple real-time fluorescence quantitative PCR batch reproducibility test

[0091]

[0092] Example 5

[0093] Specificity test of triple real-time fluorescence quantitative PCR

[0094] The genomic DNA (cDNA) of PDCoV, PEDV, TGEV, Escherichia coli, Clostridium perfringens, Toxoplasma gondii, Cryptosporidium, Trichomonas, Lawsonia intracellularis, Salmonella, and Isospora suis stored in the laboratory was used as templates to conduct the test according to the triple real-time quantitative PCR operation procedure, with 3 replicates for each setting and 3 negative samples at the same time. Figure 5 , 6 As shown in Figures 7 and 8, the results showed that only Lawsonia intracellularis, Salmonella and Isospora suis could amplify the fluorescence curve and CT value, indicating that this method can specifically detect Lawsonia intracellularis, Salmonella and Isospora suis.

[0095] Comparative Example 1

[0096] Specific screening of primers for Lawsonia intracellularis;

[0097] Three pairs of primers were designed for the conserved nucleotide sequence dnaA fragment of Lawsonia intracellularis. SYBR Green QPCR was used to select primers with target fragment amplification and no nonspecific curves and primer dimers. Figures 9 to 11 As shown, the first pair of primers ( Fig. 9 ) and the third pair of primers ( Fig.11 ) showed nonspecific curves and primer dimer peaks in the melting curves, while the second pair of primers ( Fig.10 ) had a specific and single melting curve, with no nonspecific curve or primer dimer peak, so the second pair of primers was selected as the primers for real-time fluorescence quantitative PCR of intracellular Lawsonia intracellularis.

[0098] refer to Figures 9 to 11 , Fig.10 For SEQ ID NO.1 to SEQ ID NO.2 of the present invention;

[0099] Fig. 9 The primers represented Fig.11 The primers represented are referenced in Table 5;

[0100] Table 5 Sequence list of two other primer sets for Lawsonia intracellularis

[0101]

[0102] Comparative Example 2

[0103] Specificity screening of primers for Salmonella;

[0104] Three pairs of primers were designed for the conserved nucleotide sequence INVA fragment of Salmonella. SYBR Green QPCR was used to select primers that amplified the target fragment and had no nonspecific curves and primer dimers. The results are shown in the figure. Figure 12 to Figure 14 As shown, the second pair of primers ( Fig.13 ) and the third pair of primers ( Fig.14 ) showed nonspecific curves and primer dimer peaks in their melting curves, while the first pair of primers ( Fig.12 ) has a specific and single melting curve, without nonspecific curves or primer dimer peaks, so the first pair of primers was selected as the primers for real-time fluorescence quantitative PCR of Salmonella.

[0105] refer to Figure 13 to Figure 14 , Fig.14 For SEQ ID NO.4 to SEQ ID NO.5 of the present invention;

[0106] Fig.13 The primers represented Fig.14 The primers represented are referenced in Table 6;

[0107] Table 6 Sequence list of two other primer sets for Salmonella

[0108]

[0109]

[0110] Comparative Example 3

[0111] Specificity screening of primers for Isospora suis;

[0112] Three pairs of primers were designed for the conserved nucleotide sequence ITS2 fragment of Isospora suis. SYBR Green QPCR was used to select primers that amplified the target fragment and had no nonspecific curves or primer dimers. The results are shown in the figure. Figures 15 to 17 As shown, the first pair of primers ( Fig.15 ) and the third pair of primers ( Fig.17 ) showed nonspecific curves and primer dimer peaks in the melting curves, while the second pair of primers ( Fig.16 ) had a specific and single melting curve, with no nonspecific curve or primer dimer peak, so the second pair of primers was selected as the primers for fluorescent quantitative PCR of Isospora suis.

[0113] refer to Figures 15 to 17 , Fig.17 For SEQ ID NO.7 to SEQ ID NO.8 of the present invention;

[0114] Fig.15 The primers represented Fig.16 The primers represented are referenced in Table 7;

[0115] Table 7 Sequence table of two other primer sets of Isospora suis

[0116]

[0117] Comparative Example 4

[0118] In this comparative example, the primers of SEQ ID NO.1 to SEQ ID NO.2, SEQ ID NO.4 to SEQ ID NO.5, and SEQ ID NO.7 to SEQ ID NO.8 of the present invention were applied to SYBR Green QPCR for verification to determine whether the three sets of primers would cause the appearance of dimers;

[0119] refer to Fig.18 It was found through experiments that it is difficult to avoid the generation of primer dimers when three pairs of primers are in the same system. However, in order to reduce the impact caused by primer dimers, the concentrations (ratios) of the three pairs of primers were optimized using the matrix method in the subsequent TaqMan QPCR test (Example 2).

[0120] Comparative Example 5

[0121] After screening the above comparative examples 1-4, the selection of three pairs of primers was determined. However, as is well known, the selection of probes has a significant impact on the detection results.

[0122] In the preliminary experiments of the present invention, we constructed many primers and probes to construct TaqMan QPCR triple detection, but without exception, the problem of Isospora suis not being successfully detected occurred.

[0123] In order to distinguish which detection primers and probes of Lawsonia intracellularis and Salmonella have an impact on the detection of Isospora suis, we conducted the following experiments:

[0124] first step

[0125] In the preliminary experiments, we selected three sets of primers and probes with good detection effects in a single state to detect Lawsonia intracellularis, Salmonella, and Isospora suis respectively; we found that each of them performed very well and could present a typical "S" curve; specifically, Fig.19 , Fig. 20 and Fig.21 ; Fig.19 is the LI single-plex TaqMan QPCR amplification curve; Fig. 20 is the SE single-plex TaqMan QPCR amplification curve; Fig.21 This is the IS single-plex TaqMan QPCR amplification curve.

[0126] The sequences of the above three sets of primers and probes are referenced in Table 8;

[0127] Table 8 Primers and probes for single detection of intracellular Lawsonia spp., Salmonella spp., and Isospora suis

[0128]

[0129] Step 2

[0130] The first step has confirmed that all three can form a good detection curve. In order to find out the influencing factors, the primers and probes of Lawsonia intracellularis and Isospora suis were used to form a TaqMan QPCR double detection system, and the primers and probes of Salmonella and Isospora suis were used to form a TaqMan QPCR double detection system.

[0131] The two detection systems were applied separately to observe the curve shapes of different objects;

[0132] refer to Fig. 22 , Fig.23 , Fig. 22 The amplification curve of Lawsonia intracellularis in the dual system showed a typical "S" curve. Fig.23The amplification curve of Isospora suis was abnormal and the fluorescence signal intensity was low;

[0133] refer to Fig.24 , Fig.25 , Fig.24 The amplification curve of Salmonella in the dual system showed a typical "S" curve. Fig.25 The amplification curve of Isospora suis was abnormal and the fluorescence signal intensity was low;

[0134] The above results show that the detection of Lawsonia intracellularis and Salmonella is not affected no matter what state they are in, while the detection of Isospora suis is more susceptible to the influence of external primers and probes;

[0135] After screening a large number of primers in Comparative Example 2, we believe that optimizing the probe may be the only way to solve this problem. We speculate that the probe may be a very core factor affecting the accuracy and effectiveness of the final multiplex detection; in order to solve this problem, we continue to further optimize the probe.

[0136] Step 3

[0137] In this step, referring to Table 9, we experimented with 2 groups of probes for Isospora suis, and finally screened and obtained the probe of the present invention.

[0138] Table 9 Probe list of Isospora suis

[0139]

[0140] The relevant sequences and detection results can be found in Example 1.

[0141] The above experiments can confirm that the accuracy, sensitivity and specificity of triple real-time fluorescence quantitative PCR detection of intracellular Lawsonia intracellularis, Salmonella and Isospora suis are not only related to the primers, but also closely related to the probes. In particular, there are few existing detection methods for Isospora suis. The applicant finally determined the key influencing factor as the probe for Isospora suis detection through repeated verification and combined with the optimal primer combination, and screened out an optimal probe sequence to achieve the purpose of the present invention.

Claims

1. A primer set and probe for triple real-time fluorescence quantitative PCR detection of Lawsonia intracellularis, Salmonella, and Isospora suis, characterized in that: The sequences of primers and probes for specific amplification of Lawsonia intracellularis are shown in SEQ ID NO.1 to SEQ ID NO.3; The sequences of primers and probes for specific amplification of Salmonella are shown in SEQ ID NO.4 to SEQ ID NO.6; The sequences of primers and probes for specific amplification of Isospora suis are shown in SEQ ID NO.7 to SEQ ID NO.

9.

2. A triple real-time fluorescence quantitative PCR detection kit for detecting Lawsonia intracellularis, Salmonella, and Isospora suis, characterized in that: The detection kit comprises the primer set and probe as claimed in claim 1 and 2×AceQUniversal U + Probe Master Mix V2, ddH2O.

3. The triple real-time fluorescence quantitative PCR detection kit according to claim 2, characterized in that: 2×AceQ Universal U + Probe Master Mix V2 totaling 10 μL; 1 μL of each of the upstream primer and the downstream primer of the primers with a concentration of 10 μM for specific amplification of Lawsonia intracellularis; 0.2 μL of the probe that specifically amplifies Lawsonia intracellularis at a concentration of 10 μM; 0.8 μL of each of the upstream primer and the downstream primer of the primers with a concentration of 10 μM for specific expansion of Salmonella; The probe for specific amplification of Salmonella at a concentration of 10 μM was 0.3 μL; 0.2 μL of each of the upstream primer and the downstream primer of the primers with a concentration of 10 μM for specific amplification of Isospora suis; The probe that specifically amplifies Isospora suis at a concentration of 10 μM was 0.3 μL.

4. The triple real-time fluorescence quantitative PCR detection kit according to claim 2, characterized in that: The reaction conditions applicable to the detection kit are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 10 seconds, annealing and extension at 55°C for 30 seconds, for a total of 45 cycles.

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