CN110257544B - Ergota germ fluorescent quantitative PCR detection reagent, detection kit and application - Google Patents
Ergota germ fluorescent quantitative PCR detection reagent, detection kit and application Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR detection reagent for ergot germ, a detection kit and application. The invention provides a complete set of single-stranded DNA for detecting ergot germ, which consists of a primer pair and a single-stranded DNA probe; the primer pair consists of single-stranded DNA shown in SEQ ID No.1 and single-stranded DNA shown in SEQ ID No. 2; the nucleotide sequence of the single-stranded DNA probe is SEQ ID No.3. The invention combines the PCR technology and the fluorescence detection, constructs and screens a pair of primers and a probe with high amplification efficiency and good specificity, overcomes the defects of time consumption, easy pollution, requirement of electrophoresis detection after amplification and the like of the conventional PCR, can carry out quick and accurate qualitative and quantitative detection on the ergot germs in the sample, and has the advantages of simplicity, easy operation, intuitive result, high sensitivity, good repeatability and the like.
Description
Technical Field
The invention relates to the field of detection of pathogenic fungi, in particular to a fluorescent quantitative PCR detection reagent for ergot bacteria, a detection kit and application.
Background
Claviceps purpurea (Claviceps purpurea) belongs to the genus Claviceps, clavicipitaceae, ascomycotina, pyrenomycetes, sphaeriales. It is parasitic in ovary of gramineous plants such as rye, wheat, barley, oat, etc., and changes ovary into sclerotium, which is called ergot because it is shaped like wheat grain. Ergot bacteria can infect many cereal grain plants and grassy weeds.
On one hand, the ergot caused by the ergot germ is an important disease of wheat, so that the yield of crops is greatly reduced, the yield loss of rye is generally 5%, and the yield loss of wheat is generally 10%. Economic losses also include the inability or reduced grade of the contaminated grain. On the other hand, ergot contains various toxic ergot alkaloids such as ergotamine and ergoline. People and livestock are poisoned by eating it by mistake, for example, the appetite of cattle can be reduced and the weight of cattle can be reduced by containing 0.5 percent of ergot (weight ratio) in the food.
In recent years, along with the development of import and export trade, the import of soybeans in China is continuously increased, and only 2016 years are used for importing soybeans 8391 for ten thousand. While imported soybeans are increased continuously, a plurality of pests come along with the imported soybeans, and rat-dung-like black substances, most of which are sclerotium of soybean sclerotinia sclerotiorum, are often detected in the process of quarantine of the imported soybeans. However, grassy weeds such as wheat, barley, rye grass and brome are often mixed in imported soybeans, the possibility that black substances are ergot is high, and ergot germs are captured from soybeans in the united states and brazil many times in many ports in China. Because sclerotia of the soybean sclerotinia sclerotiorum is similar to the ergot in shape, and the ergot is infected by different host wheat, the infection environment and other factors are changed, the formed sclerotia have different shapes, and the ergot can be broken due to the mechanical action in the harvesting and processing processes, so the change in shape is larger. Ergot cannot be detected in rice, corn and beans according to the regulation of GB 2715-2016 national food safety Standard. GB 19641-2015 "plant oil sanitation Standard" stipulates that ergot/% < 0.05% "is contained in rapeseed, and ergot cannot be detected in other oil materials. Therefore, the method has important significance for detecting and identifying the hybrid ergot in the soybean.
The detection method of ergot (Claviceps purpurea) mainly comprises a traditional sensory alkaloid color development method, a serological method, a molecular biological method and the like. GB/T5009.36-2003, analytical methods for food sanitation standards, GB 19641-2015, plant oil sanitation standards and GB 2715-2016, national food safety standards, adopt methods for qualitative detection of suspected substances, namely, ergorubin and ergot alkaloid. However, the method needs a large amount of samples, the process is complex, the color development phenomenon is not obvious in the actual operation, and the amount of the samples found in imported food and oil crops is small, so that the quarantine identification difficulty is further increased. The enzyme-linked immunosorbent assay (ELISA) has the characteristics of short detection time, strong specificity and simple pretreatment of instrument equipment and samples, is suitable for screening and field detection of large-batch samples, and has the defects of limited preservation time and large dosage of enzyme-labeled antibodies, cross reaction, possibility of false positive and false negative and the like; and (3) ribosome DNA sequence analysis, namely amplifying the extracted DNA by using a fungus ITS universal primer, and carrying out sequence analysis after sequencing an amplification product so as to determine whether the DNA is ergot germ. Although the amount of the obtained biological information is large, the experimental operation is complicated, and the detection period is long; the techniques such as Restriction Fragment Length Polymorphism (RFLP), random amplified DN A polymorphism (RAPD), pulse zone electrophoresis (PFGE) and the like are used for describing the molecular characteristics of ergot strains, identifying the strains and providing a basis for establishing subspecies classification. Many complicated procedures in sequence analysis can be omitted, but multiple enzyme digestion is required, time and labor are wasted, and the molecular detection methods are subjected to PCR (polymerase chain reaction) post-treatment such as electrophoresis and the like, contact with a toxic reagent ethidium bromide and are easy to generate cross contamination to cause false positive results. The Loop-mediated isothermal amplification (LAMP) technology has the advantages of high sensitivity, fast reaction and simple operation, but the problem of false positive is serious because the sensitivity is high and aerosol pollution is easily formed once the cover is opened.
The fluorescent quantitative PCR (Real-time fluorescent PCR) has the advantages of short detection period, high specificity and sensitivity, no need of PCR post-treatment and the like. And the Ct value has a strict linear relation with the logarithm value of the initial template amount in the reaction system. In addition to the qualitative determination of whether the reaction system contains the target DNA, the relative quantification of the template DNA in the reaction system can be performed. The Tm value of the ordinary TaqMan probe is 65-72 ℃, the fragment length is 25-40 nt, and due to the characteristic of high specificity, if the probe is not completely matched with a target gene sequence, the problems of low detection efficiency and even no generation of a fluorescent signal can occur. TaqMan MGB (Minor Groove Binder) probe technology. The probe has MGB molecules at the 3' end, so that the Tm value of the probe is improved, the length of the probe is shortened, and the reaction conditions of the whole fluorescence PCR are met.
Therefore, the research and establishment of the specific and sensitive fluorescent quantitative PCR detection method capable of directly detecting the low-content ergot (C.purpurea) has important practical application value in the aspects of quarantine, diagnosis and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a fluorescent quantitative PCR detection reagent which has high specificity and strong sensitivity on ergot germ and can carry out quantitative and rapid detection.
In a first aspect, the invention claims a set of single stranded DNA for use in the detection of ergot bacteria.
The invention claims a set of single-stranded DNA for detecting ergot germ, which consists of a primer pair and a single-stranded DNA probe.
Wherein, the primer pair can be any one of the following:
(a1) A primer pair consisting of a single-stranded DNA shown in SEQ ID No.1 and a single-stranded DNA shown in SEQ ID No. 2;
(a2) And (b) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.1 and SEQ ID No.2, and has the same functions as the primer pair in (a 1).
The nucleotide sequence of the single-stranded DNA probe is SEQ ID No.3.
Furthermore, the 5 'end of the single-stranded DNA probe is marked with a fluorescence reporter group, and the 3' end of the single-stranded DNA probe is marked with a fluorescence quenching group.
Still further, the fluorescent reporter group can be FAM; the fluorescence quenching group can be TAMRA. The fluorescent reporter group FAM may be replaced by a luminescent group such as TET, VIC, JOE, etc.
In a second aspect, the invention claims a primer pair for detecting ergot bacteria.
The primer pairs claimed in the present invention are those described in the first aspect hereinbefore.
In a third aspect, the invention claims reagents for detecting ergot bacteria.
Besides the complete set of single-stranded DNA or the primer, the reagent for detecting the ergot bacteria, which is claimed by the invention, can also contain TaqMan real-time fluorescent quantitative PCR amplification buffer solution and dd H 2 O。
In a fourth aspect, the invention claims a kit for detecting ergot bacteria.
The claimed kit for detecting ergot bacteria of the present invention contains the following components (10. Mu.L reaction X100 times):
solution I:
50mL of Universal PCR Master Mix (e.g., product of Thermo Fisher Scientific, cat. No. 4324018);
solution II: 20 mu L of primer solution; in the primer solution, the concentrations of the single-stranded DNA shown in SEQ ID No.1 and the single-stranded DNA shown in SEQ ID No.2 are both 10 mu mol/L;
solution III: 70 mu L of probe solution; in the probe solution, the concentration of the single-stranded DNA shown in SEQ ID No.3 is 10 mu mol/L;
solution IV: 1.25mL of sterilized deionized water, multiplied by 2;
solution V: positive control solution 100 μ L; the positive control solution contains ergot germ genome DNA;
solution VI: negative control solution 100. Mu.L; the negative control solution does not contain ergot germ genome DNA, and specifically can be a solution containing sclerotinia sclerotiorum genome DNA;
solution VII: quantifying 100 mu L of standard solution; the quantitative standard solution contains ergot germ genome DNA with the concentration of 100 ng/. Mu.L.
In a fifth aspect, the present invention claims a method for detecting ergot bacteria in a sample to be tested.
The method for detecting the ergot bacteria in the sample to be detected claimed in the present invention can be (b 1) or (b 2) as follows:
(b1) The method comprises the following steps: extracting total DNA from a sample to be detected as a template, and carrying out PCR amplification by the primer pair to obtain an amplification product; and then determining whether the sample to be detected contains ergot bacteria or not according to the following steps: if the amplification product contains a 59bp DNA fragment, the sample to be detected contains or is candidate to contain Claviceps purpurea; if the amplification product does not contain the DNA fragment of 59bp, the ergot germ is not contained or does not exist in the candidate sample.
(b2) The method comprises the following steps: extracting total DNA from a sample to be detected, using the total DNA as a template, and performing fluorescent quantitative PCR amplification on the complete set of single-stranded DNA; and then determining whether the sample to be detected contains ergot bacteria or not according to the Ct value and the amplification curve as follows: if the Ct value is less than or equal to 35 and the amplification curve is positive (S-type or J-type curve), the ergot germ is contained or candidate contained in the sample to be detected (positive); if the Ct value is not present or is greater than or equal to 40 or no amplification curve is present, the ergot germ is not contained or candidate is not contained in the sample to be detected (negative).
Further, if the Ct value is greater than 35.0 and less than 40.0 and the amplification curve is an S-type or J-type curve, the sample to be detected needs to be re-detected, and the result determination method is the same as above.
In the above (b 1), the 59bp DNA fragment is specifically a DNA fragment represented by SEQ ID No. 4.
In a sixth aspect, the invention claims a method for detecting the content of genomic DNA of Clavicepidopsis in a sample to be detected.
The method for detecting the content of the ergot germ genome DNA in the sample to be detected provided by the invention can comprise the following steps:
(c1) The quantitative standard solution in the kit described above was diluted to a series of known concentrations and used as templates (e.g., 10-fold serial dilutions were performed to obtain 10 －1 ～10 －7 Double-diluted DNA template), performing fluorescent quantitative PCR amplification on the complete set of single-stranded DNA as described above, and drawing a fluorescent quantitative PCR standard curve;
such as: and taking the logarithm value of the template concentration as an X axis and the Ct value as a Y axis to make a regression curve and obtain a regression equation. According to the result conversion, the genomic DNA content of the ergot germ can be obtained from the Ct value of the unknown sample.
(c2) Extracting total DNA from a sample to be detected, using the total DNA as a template, and performing fluorescent quantitative PCR amplification on the complete set of single-stranded DNA; and (c) obtaining the content of the ergot germ genome DNA in the sample to be detected according to the fluorescent quantitative PCR standard curve drawn in the step (c 1).
In the foregoing fifth and sixth aspects, when the fluorescent quantitative PCR amplification is performed, both the single-stranded DNAs constituting the primer pair may be present in the reaction system at a concentration of 0.2. Mu. Mol/L, and the single-stranded DNA probe may be present in the reaction system at a concentration of 0.7. Mu. Mol/L.
Specifically, 10. Mu.L of the reaction system: 5 μ L
Universal PCR Master Mix, 0.2. Mu.L CP-F (10. Mu. Mol/L), 0.2. Mu.L CP-R (10. Mu. Mol/L), 0.7. Mu.L CP-P (10. Mu. Mol/L), 1. Mu.L of DNA to be tested, was supplemented to 10. Mu.L with sterile deionized water. Wherein CP-F, CP-R and CP-P are three single-stranded DNAs (primers or probes) shown in SEQ ID Nos. 1-3, respectively.
In the foregoing fifth and sixth aspects, the fluorescent quantitative PCR amplification is performed at an annealing temperature of 60 ℃. The method specifically comprises the following steps: pre-denaturation at 95 ℃ for 10min;95 ℃ 15s,60 ℃ 1min,40 cycles.
In the fifth and sixth aspects of the present invention, in a specific embodiment of the present invention, the sample to be tested is specifically ergot (c. Purpurea) or Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) or rhododendron wilt (ovulina azalea) or camellia rot (ciboria camellia sinensis).
In a seventh aspect, the invention claims any of the following applications:
(d1) Use of a set of single-stranded DNA as hereinbefore described or a pair of primers as hereinbefore described in the preparation of a reagent as hereinbefore described;
(d2) Use of a set of single-stranded DNA as hereinbefore described or a primer pair as hereinbefore described or an agent as hereinbefore described in the preparation of a kit as hereinbefore described;
(d3) Use of the set of single-stranded DNA as described above or of the primer pair as described above or of the reagent as described above or of the kit as described above for the detection of ergot bacteria or for the preparation of a product for the detection of ergot bacteria.
In the invention, the ergot bacteria is specifically ergot bacteria (c.
The ergot germ fluorescence quantitative PCR detection reagent disclosed by the invention is used for combining a PCR technology with fluorescence detection, constructing and screening a pair of primers and a probe with good amplification efficiency and specificity, overcoming the defects of time consumption, easiness in pollution, requirement of electrophoresis detection after amplification and the like of the conventional PCR, being capable of quickly and accurately performing qualitative and quantitative detection on the ergot germ in a sample, and having the advantages of simplicity, easiness in operation, visual result, high sensitivity, good repeatability and the like. The method has the following specific advantages:
1. compared with the conventional PCR detection reagent, the kit has the advantages of specificity and sensitivity; the method can accurately detect the ergot germ with low content in the sample, and is a method very suitable for the ergot germ detection.
2. Compared with the conventional PCR detection reagent, the invention has the advantages of wide application range and safe use; the ergot germ can be quickly detected, and the application range is wide; the invention overcomes the defects that the products of the conventional PCR need electrophoresis detection and contact with toxic reagents, reduces the biological potential safety hazard and improves the use safety.
3. Compared with the conventional PCR detection reagent, the invention has the advantages of large detection amount and rapid detection; can simultaneously detect a large number of samples, has high flux and can reduce the risk of DNA or PCR product pollution; the detection time of the conventional PCR is generally more than 6 hours, the invention does not need electrophoretic analysis after amplification, and the detection time of the sample is within 4 hours.
4. Compared with the conventional PCR, the fluorescent quantitative PCR makes a quantitative and objective estimation and judges positive, negative and suspicious results. More importantly, the fluorescent quantitative PCR is particularly suitable for the rapid detection of germs in samples which are small in sample amount and difficult to separate and culture.
5. The reagent kit can make qualitative and quantitative detection results within 4 hours after receiving the sample. The fluorescent quantitative PCR kit is a specific, sensitive and stable detection method for detecting the ergot germ.
Drawings
FIG. 1 shows the results of the primer optimization experiments for Claviceps purpurea fluorescent quantitative PCR.
FIG. 2 shows the results of the probe optimization assay of Claviceps purpurea fluorescent quantitative PCR.
FIG. 3 shows the result of the specificity test of Claviceps purpura fluorescent quantitative PCR.
FIG. 4 shows the results of the sensitivity test of Claviceps purpura fluorescent quantitative PCR.
FIG. 5 is a standard curve for Claviceps purpura fluorescent quantitative PCR.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of fluorescent quantitative PCR kit for detection of Claviper bacteria
1. Design of primers and probes
The invention selects a conservative fragment of an Ergota germ translation Elongation factor (Elongation factor 1 alpha, EF-1 alpha) region as a target, performs homology analysis and comparison through Ergota germ (C.purpurea) and an approximate variety EF-1 alpha sequence reported in GenBank, selects a conservative fragment (59 bp) of the Ergota germ EF-1 alpha region, and applies primer expression 5.0 software to design a primer and a probe. The primer and the probe are synthesized by adopting a beta-acetonitrile ammonium phosphite chemical synthesis method, an automatic DNA synthesizer is used for synthesizing the OligoDNA probe, the two ends of the OligoDNA probe are fluorescently labeled during synthesis, a fluorescent reporter group labeled at the 5 'end of the probe is FAM, and a quenching group TAMRA which does not fluoresce is labeled at the 3' end of the OligoDNA probe. After carrying out optimal pairing screening experiments on a plurality of pairs of designed and synthesized primers and probes, determining a pair of primers and a probe with the best amplification efficiency and specificity, wherein the primer sequences are respectively shown as SEQ ID No.1 and SEQ ID No.2, the probe sequence is shown as SEQ ID No.3, the length of an amplified target fragment is 59bp, and the sequence is shown as SEQ ID No. 4.
The primer sequences are as follows:
CP-F：5′-TCAACCACCCCGGTCAAG-3′(SEQ ID No.1)；
CP-R：5′-GGCAGTGTGGCAATCAAGAA-3′(SEQ ID No.2)；
specific probe sequence CP-P:5'-FAM-CTGGTTACGCCCCC-TAMRA-3' (SEQ ID No. 3).
2. The components of the standardized kit (10. Mu.L reactions X100 times) are shown in Table 1.
TABLE 1 standardization kit Components (10. Mu.L reactions X100 times)
Note:
universal PCR Master Mix is a product of Thermo Fisher Scientific, inc. under the product number 4324018.
Example 2 fluorescent quantitative PCR detection of Ergota
1. Detection method and result judgment standard
1. PCR amplification of purpeu fluorescence
The method is characterized in that a 10-microliter volume reaction system is adopted for the purpeu fluorescent quantitative PCR, and in an ABI7900 type fluorescent quantitative PCR instrument, the reaction system is as follows: 5 μ L
Universal PCR Master Mix, 0.2. Mu.L CP-F (10. Mu. Mol/L), 0.2. Mu.L CP-R (10. Mu. Mol/L), 0.7. Mu.L CP-P (10. Mu. Mol/L), 1. Mu.L genomic DNA, supplemented to 10. Mu.L with sterile deionized water. Equal volumes of water were used instead of genomic DNA as a blank. Meanwhile, a positive control and a negative control 10 mu L reaction system 2 tube are arranged.
The PCR reaction was carried out with the following reaction parameters: preheating at 50 deg.C for 2min; pre-denaturation at 95 ℃ for 10min;95 ℃ 15s,60 ℃ 1min,40 cycles.
And (3) carrying out accurate screening on the concentrations of the screened primers and probes by using the positive ergot germ genome DNA and carrying out an optimal pairing screening experiment on a plurality of pairs of primers and probes designed according to the reaction system and the reaction parameters so as to obtain a lowest Ct value and a higher fluorescence intensity increase value (delta Rn). At each test, each sample test was performed in 3-tube parallel. When the negative control is negative and the positive control is positive, the whole test is effective, and the result shows that the pair of specific primers and the specific fluorescent probe have low Ct value and high sensitivity and are suitable primers and probes. Multiple copies of each sample were made for stability and reproducibility testing.
Wherein, the screening of the primer, the probe and the concentration thereof:
and selecting the optimal concentration ratio of the primer, the probe and the template to obtain the lowest Ct value and the highest Delta Rn of the fluorescent quantitative PCR reaction, thereby improving the amplification efficiency and sensitivity. The primer concentration is increased from 0.1 mu mol/L to 1 mu mol/L by 0.1 mu mol/L to determine the optimal final concentration of the primer. The concentration of the probe is increased from 0.1 mu mol/L to 1 mu mol/L by 0.1 mu mol/L, and the optimal final concentration of the probe is determined.
As shown in FIGS. 1 and 2, the amplification effect was the best when the concentration of the upstream and downstream primers was 0.2. Mu. Mol/L; the amplification effect was the best at a probe concentration of 0.7. Mu. Mol/L.
2. Preparation of ergot germ quantitative standard solution, establishment of standard curve and calculation of content of ergot germ in sample
According to
The DNA of Clavicepina Clavicipiens was extracted with the Plant DNA Auto Kit, and its OD was measured with a spectrophotometer 260 Value to perform DNA template quantification, OD 260 A value of 1 corresponds to 50. Mu.g/mL of double-stranded DNA. The extracted DNA was diluted and quantified to 100 ng/. Mu.L using sterile deionized water. The standard was serially diluted 10-fold to give 10 －1 ～10 －7 The DNA template diluted by times is used for fluorescent quantitative PCR amplification, and a regression curve is drawn by taking the logarithm value of the template concentration as an X axis and the Ct value as a Y axis to obtain a regression equation. According to the result conversion, the Ct value of the unknown sample can be obtainedContains the DNA content of Claviceps purpurea.
3. Setting of conditions for analysis of results
The threshold line setting principle is adjusted according to the noise condition of the instrument, and the condition that the threshold line just exceeds the highest point of the amplification curve of the normal negative sample is taken as the standard.
4. Quality control standard
Negative control has no Ct value and no typical amplification curve.
The Ct value of the positive control should be less than 35.0 and a typical positive amplification curve (S-or J-type amplification curve) appears.
-quantitative standard amplification curve: a typical amplification curve appears, the exponential region is obvious, 7 points have good linear range, the platform regions are converged together, and the correlation coefficient is above 0.99.
5. Result description and determination
Negative
And if the Ct value is not greater than 40, the ergot germ does not exist in the sample.
Positive for
Ct value should be less than or equal to 35.0, and a typical positive amplification curve (S-type or J-type amplification curve) appears, indicating the presence of ergot bacteria in the sample.
-principle of validity
Ct values ranged from 35.0 to 40.0 (35 and 40 excluded), and samples with typical positive amplification curves (S-or J-type amplification curves) were suggested for redo. And (5) redoing the result and judging the result as above.
-quantifying: and comparing the regression curve of the sample DNA with the regression curve according to the standard product to make the quantification of the sample DNA.
2. Specificity test of Purpurea fluorescent quantitative PCR
The sclerotia on different plant products are selected for specific comparison detection, including sclerotia of soybean sclerotinia sclerotiorum and ergot detected from soybean and barley. A sample to be detected: SC1 (ergot c. Purpurea was isolated from barley), SC2 (ergot c. Purpurea was isolated from barley), SC3 (ergot c. Purpurea was isolated from barley), SC4 (ergot c. Purpurea was isolated from soybean), SC5 (ergot c. Purpurea was isolated from soybean), SC6 (ergot c. Purpurea was isolated from soybean), SC7 (Sclerotinia sclerotiorum was isolated from soybean), SC8 (Sclerotinia sclerotiorum was isolated from soybean), SC9 (rhododendron wilt ovuligina azalea was isolated from rhododendron), SC10 (camellia saprophyta camelliae was isolated from camellia.
1. Extracting the genome DNA of a sample to be detected;
2. and (3) performing real-time fluorescence PCR by using the genomic DNA obtained in the step (1) as a template. Reaction system 10. Mu.L: 5 μ L
Universal PCR Master Mix, 0.2. Mu.L CP-F (10. Mu. Mol/L), 0.2. Mu.L CP-R (10. Mu. Mol/L), 0.7. Mu.L CP-P (10. Mu. Mol/L), 1. Mu.L genomic DNA, was supplemented to 10. Mu.L with sterile deionized water. In the reaction system, the concentration of CP-F is 0.2 mu mol/L, the concentration of CP-R is 0.2 mu mol/L, and the concentration of CP-P is 0.7 mu mol/L. Equal volumes of water were used instead of genomic DNA as a blank. Reaction parameters are as follows: pre-denaturation at 95 ℃ for 10min; 15s at 95 ℃, 1min at 60 ℃,40 cycles;
the results are shown in FIG. 3. It is shown that only when c.purpurea is used as the sample to be tested (SC 1 to SC 6), a positive amplification curve is displayed, and no other sample to be tested shows a positive amplification curve. The complete set of reagents for detecting ergot bacteria have strong specificity.
3. Sensitivity test of PCR by purpeuea fluorescence quantification
1. And taking the DNA of the SC1 in the step two as a template, and performing gradient dilution on the genomic DNA by using sterilized deionized water to serve as the template.
2. And (3) performing real-time fluorescence PCR by using the genomic DNA obtained in the step (1) as a template. Reaction system 10. Mu.L: 5 μ L
Universal PCR Master Mix, 0.2. Mu.L CP-F (10. Mu. Mol/L), 0.2. Mu.L CP-R (10. Mu. Mol/L), 0.7. Mu.L CP-P (10. Mu. Mol/L), 1. Mu.L genomic DNA, was supplemented to 10. Mu.L with sterile deionized water. In the reaction system, the concentration of CP-F is 0.2 mu mol/L, and CP-RThe concentration was 0.2. Mu. Mol/L and the CP-P concentration was 0.7. Mu. Mol/L. Equal volumes of water were used instead of genomic DNA as a blank.
The total number of the reaction systems is 8, and in the reaction system 1, the DNA content of the template is 30ng; in the reaction system 2, the DNA content of the template is 3ng; in the reaction system 3, the DNA content of the template is 300pg; in the reaction system 4, the DNA content of the template is 30pg; in the reaction system 5, the DNA content of the template is 3pg; in the reaction system 6, the DNA content of the template is 300fg; in the reaction system 7, the DNA content of the template was 30fg; in the reaction system 8, the DNA content of the template is 3fg; the laboratory was set up for 3 replicates.
Reaction parameters are as follows: pre-denaturation at 95 ℃ for 10min; 15s at 95 ℃, 1min at 60 ℃,40 cycles;
the results are shown in FIG. 4. The positive amplification curves are shown in the reaction systems 1-5, and no positive amplification curve exists in the reaction systems 6, 7 and 8 and a blank control, which shows that the lowest detection limit of the complete set of reagents for detecting ergot bacteria is a 3pg template DNA/10 mu L system.
4. Standard curve of purpeuea fluorescent quantitative PCR
1. And taking the DNA of the SC1 in the step two as a template, and performing gradient dilution on the genomic DNA by using sterilized deionized water to serve as the template.
2. And (3) performing real-time fluorescence PCR by using the genomic DNA obtained in the step (1) as a template. Reaction system 10 μ L:5 μ L
Universal PCR Master Mix, 0.2. Mu.L CP-F (10. Mu. Mol/L), 0.2. Mu.L CP-R (10. Mu. Mol/L), 0.7. Mu.L CP-P (10. Mu. Mol/L), 1. Mu.L genomic DNA, was supplemented to 10. Mu.L with sterile deionized water. In the reaction system, the concentration of CP-F is 0.2 mu mol/L, the concentration of CP-R is 0.2 mu mol/L, and the concentration of CP-P is 0.7 mu mol/L. Equal volumes of water were used instead of genomic DNA as a blank.
The total number of the reaction systems is 5, and in the reaction system 1, the DNA content of the template is 30ng; in the reaction system 2, the DNA content of the template is 3ng; in the reaction system 3, the DNA content of the template is 300pg; in the reaction system 4, the DNA content of the template is 30pg; in the reaction system 5, the DNA content of the template is 3pg; the laboratory was set up for 3 replicates.
Reaction parameters are as follows: pre-denaturation at 95 ℃ for 10min; 15s at 95 ℃, 1min at 60 ℃,40 cycles;
the amplification results are shown in FIG. 4. The real-time fluorescence PCR detection of the total DNA with known concentration gradient shows that the higher the template concentration is, the smaller the Ct value is, and 3 parallel test results show that the DNA template concentration is extremely related to the Ct value. y = -3.78x +21.17 (R) 2 = 0.9960), x represents the DNA template concentration and y represents the corresponding Ct value, and the standard curve is shown in fig. 5.
5. Stability and repeatability test of purpeu fluorescent quantitative PCR
When the reproducibility and the stability of the method for detecting the ergot germ by the fluorescent quantitative PCR are evaluated, the positive samples and the negative samples are used for repeatedly carrying out the fluorescent quantitative PCR detection under the same reaction condition according to the step I, and each sample of each test is made into 6 parallel reaction tubes and is repeated for 12 times. The conclusion is that: the pair of specific primers and the specific fluorescent probe have good stability and repeatability.
<110> Ningbo inspection and quarantine scientific and technical research institute
<120> ergot germ fluorescence quantitative PCR detection reagent, detection kit and application
<130> GNCLN191146
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
<400> 1
tcaaccaccc cggtcaag 18
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 14
<212> DNA
<213> Artificial sequence
<400> 3
ctggttacgc cccc 14
<210> 4
<211> 59
<212> DNA
<213> Artificial sequence
<400> 4
tcaaccaccc cggtcaagtc ggtgctggtt acgcccccgt tcttgattgc cacactgcc 59
Claims (10)
1. The complete set of single-stranded DNA for detecting the ergot germ consists of a primer pair and a single-stranded DNA probe;
the primer pair is composed of a single-stranded DNA shown in SEQ ID No.1 and a single-stranded DNA shown in SEQ ID No. 2;
the nucleotide sequence of the single-stranded DNA probe is SEQ ID No.3.
2. The set of single-stranded DNA of claim 1, wherein: the 5 'end of the single-stranded DNA probe is marked with a fluorescent reporter group, and the 3' end of the single-stranded DNA probe is marked with a fluorescent quenching group.
3. The set of single-stranded DNA of claim 2, wherein: the fluorescent reporter group is FAM; the fluorescence quenching group is TAMRA.
4. A reagent for detecting ergot bacteria, characterized in that: the reagent contains TaqMan real-time fluorescent quantitative PCR amplification buffer solution and dd H in addition to the complete set of single-stranded DNA of any one of claims 1 to 3 2 O。
5. A kit for detecting ergot bacteria, characterized in that: the kit comprises the following components:
solution I:2 XTaqMan Universal PCR Master Mix;
solution II: a primer solution; in the primer solution, the concentrations of the single-stranded DNA shown in SEQ ID No.1 and the single-stranded DNA shown in SEQ ID No.2 are both 10 mu mol/L;
solution III: a probe solution; in the probe solution, the concentration of the single-stranded DNA shown in SEQ ID No.3 is 10 mu mol/L;
solution IV: sterilizing deionized water;
solution V: a positive control solution; the positive control solution contains ergot germ genome DNA;
solution VI: a negative control solution; the negative control solution does not contain ergot germ genome DNA;
solution VII: quantifying a standard solution; the quantitative standard solution contains ergot germ genome DNA with the concentration of 100 ng/mu L.
6. A method for detecting whether a sample to be detected contains ergot bacteria is (b 1) or (b 2) as follows:
(b1) The method comprises the following steps: extracting total DNA from a sample to be detected, and performing PCR amplification by using the primer pair as claimed in claim 1 as a template to obtain an amplification product; and then determining whether the sample to be detected contains ergot bacteria or not according to the following steps: if the amplification product contains the DNA fragment of 59bp, the sample to be detected contains or is candidate to contain Claviper bacteria; if the amplification product does not contain the DNA fragment of 59bp, the sample to be detected does not contain or candidate does not contain ergot bacteria;
(b2) The method comprises the following steps: extracting total DNA from a sample to be tested, and performing fluorescent quantitative PCR amplification by using the set of single-stranded DNA as claimed in any one of claims 1 to 3 as a template; and then determining whether the sample to be detected contains ergot germs according to the Ct value and the amplification curve as follows: if the Ct value is less than or equal to 35 and the amplification curve is positive, the ergot germ is contained or candidate contained in the sample to be detected; if the Ct value is not present or is greater than or equal to 40 or no amplification curve is present, the ergot germ is not contained or candidate is not contained in the sample to be detected.
7. The method of claim 6, wherein: the DNA fragment of 59bp is the DNA fragment shown in SEQ ID No. 4.
8. A method for detecting the content of Claviper germ genome DNA in a sample to be detected comprises the following steps:
(c1) Diluting the quantitative standard solution in the kit of claim 5 into a series of known concentrations, respectively using the diluted quantitative standard solution as a template, performing fluorescent quantitative PCR amplification on the set of single-stranded DNA of any one of claims 1 to 3, and drawing a fluorescent quantitative PCR standard curve;
(c2) Extracting total DNA from a sample to be tested, and performing fluorescent quantitative PCR amplification by using the set of single-stranded DNA according to any one of claims 1 to 3 as a template; and (c) obtaining the content of the ergot germ genome DNA in the sample to be detected according to the fluorescent quantitative PCR standard curve drawn in the step (c 1).
9. The method according to any one of claims 6-8, wherein: when the fluorescence quantitative PCR amplification is carried out, the concentration of the two single-stranded DNA forming the primer pair in a reaction system is 0.2 mu mol/L, and the concentration of the single-stranded DNA probe in the reaction system is 0.7 mu mol/L; and/or
When the fluorescent quantitative PCR amplification is carried out, the annealing temperature is 60 ℃.
10. Any of the following applications:
(d1) Use of a set of single-stranded DNAs of any one of claims 1 to 3 for the preparation of a reagent of claim 4;
(d2) Use of a set of single-stranded DNA according to any one of claims 1 to 3 or a reagent according to claim 4 for the preparation of a kit according to claim 5;
(d3) Use of a set of single stranded DNA as claimed in any one of claims 1 to 3 or a reagent as claimed in claim 4 or a kit as claimed in claim 5 for the detection of ergot bacteria or for the preparation of a product for the detection of ergot bacteria.
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| CN105886619A (en) * | 2016-04-27 | 2016-08-24 | 张梅 | Kit and method for detecting claviceps purpurea |
Non-Patent Citations (3)
| Title |
|---|
| Characterization of Claviceps species pathogenic on sorghum by sequence analysis of the β-tubulin gene intron 3 region and EF-1α gene intron 4;Paul W. Tooley等;《Mycologia》;20011231;第93卷(第3期);第541-551页 * |
| Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR;Jeremiah K.S. Dung等;《Plant Disease》;20181231;第102卷;第2487-2493页 * |
| Sylvie Pažoutová等.Delimitation of cryptic species inside Claviceps purpurea.《Fungal Biology》.2014,第119卷 * |
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