CN103789430A - Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit - Google Patents
Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit Download PDFInfo
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Abstract
The invention discloses a kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of the kit. The kit consists of a detachable 96-pore polymerase chain reaction (PCR) tube, PCR reaction liquid, TaqDNA polymerase, standard and control, wherein the PCR reaction liquid contains a PCR buffer solution, dNTPs, MgCl2, KCl, gelatin and various detection primers; the various detection primers are respectively specific primers for eperythrozoon, leptospira and toxoplasma. The kit is applicable to detecting whether blood is infected by one or more pathogens in the eperythrozoon, leptospira and toxoplasma. The kit disclosed by the invention is rapid in detection speed, high in detection sensitivity, good in specificity, and simple and convenient to operate; and the kit can simultaneously identify and detect one or more specimen in mixed infection of the eperythrozoon, leptospira and toxoplasma, thus greatly improving detection accuracy and reduce false positive rate.
Description
Technical field
The present invention relates to biotechnology, relate in particular to the test kit of Eperythrozoon, Leptospira, toxoplasma gondii in a kind of rapid detection blood, can differentiate detection to the blood preparation of single or multiple polyinfection red cell bodies, Leptospira, toxoplasma gondii simultaneously.
Background technology
Eperythrozoon (Eperythrozoon), Leptospira (Leptospira), toxoplasma gondii (Toxoplasma gondii) be all over the world all pandemic three-type-person raise ill substance altogether, the common feature of three is to be all present in animal blood, can cause infected animal fervescence, be wherein important three kinds of pathogenic agent of the pig " high temperature high-fever syndrome " once reported, crowd can infect by contact infected animal and animal product pig blood etc.Eperythrozoon also claims Tubifex body, it is the unicellular protista of one parasitizing in people, animal erythrocyte surface, blood plasma and marrow, can cause that various animals are hot, hemolytic disease, the many animals such as people, pig, ox, sheep all can infect, each stage infection rate of pig reaches 80～90 ﹪, and people's infection positive rate can reach 86 ﹪.Leptospira can cause the leptospirosis of people and animal, be called for short Leptospirosis, in pandemic a kind of zoonosis all over the world, there is in various degree popular in the most areas of China, the cardinal symptom of ill piglet is jaundice, high fever, and sick pig is chronic carrier and excreter often, brings about great losses to livestock industry, and very large to people ' s health harm, be one of transmissible disease of China's keypoint control.Toxoplasma gondii parasitizes in cell, with blood flow, arrive the each position of whole body, destroy brain, heart, eyeground, cause the immunity degradation of body, increase Other diseases and infect probability, people can infect by contact infected animal and contaminated food products, especially very harmful to pregnant woman, can there is miscarriage, stillborn foetus or newborn child's illness, or after birth, have pathology or the deformity of eye, brain or liver.This three classes disease is not easy to distinguish in early days, diagnose difficulty large, and spread scope is wide, is easily transmitted to people with the animal product of carcass and pollution.Therefore, control the popular of this class disease and must pick up from source, control zoonosis popular in livestock and poultry, ensure food safety.
At present, mainly rely on the traditional method such as serology, cause of disease identification of morphology for Eperythrozoon, Leptospira, toxoplasma gondii, but that these detection methods have detection time is long, workload is large, program is loaded down with trivial details, be difficult to adapt to the needs of the quarantine of modernization scale raising, the quarantine of animal import and export disease, strict pathogen detection is the important step that ensures animal, human-body safety, therefore need badly set up a kind of easy and simple to handle, sensitivity and specificity is high, quick and precisely, a pipe detects multiple pathogens method.
Multiplex PCR (multiplex PCR), claim again composite PCR, it is different from normal PCR technology can only detect single target gene, it is to add multipair Auele Specific Primer in same PCR reaction system, amplify the PCR reaction of multiple nucleic acid fragments simultaneously, therefore rapid detection or evaluation when can be used for multiple-microorganism, have the features such as high efficiency, high specific, easy, quick, easy detection.This technology is mainly used in the rapid detection of various bacteria, virus disease at present, and does not have report and open to detect the multiple PCR fast detecting method of Eperythrozoon, Leptospira, toxoplasma gondii in blood simultaneously.
Summary of the invention
An object of the present invention is for the deficiencies in the prior art, the test kit of Eperythrozoon, Leptospira, toxoplasma gondii in a kind of rapid detection blood is provided.
For achieving the above object, first the present invention designs respectively 3 couples of Auele Specific Primer: F1/R1, F2/R2, F3/R3 according to the feature target gene of Eperythrozoon, Leptospira, toxoplasma gondii.Length after Eperythrozoon Auele Specific Primer F1/R1 amplification is 186bp, and the length after Leptospira Auele Specific Primer F2/R2 amplification is 293bp, and the length after toxoplasma gondii Auele Specific Primer F3/R3 amplification is 407bp.Conservative in each Auele Specific Primer has in height, plant between the advantage such as specificity.Fragment length different sizes after amplification, can separately distinguish by electrophoresis.
Test kit of the present invention is made up of detachable 96 hole PCR pipes, PCR reaction solution, Taq archaeal dna polymerase, standard substance, reference substance.
Described standard substance are three kinds of DNA samples that contain respectively Eperythrozoon, Leptospira, bow-shaped worm dna fragment;
Described reference substance is divided into positive reference substance and negative control product; Wherein positive reference substance is Eperythrozoon, Leptospira, bow-shaped worm dna sample mix liquid; Negative control product are aseptic deionized water;
Described PCR reaction solution contains 10mmol/L PCR damping fluid, 0.5mmol/L dNTPs, 1.5mmol/L MgCl
2, 50mmol/L KCl, 100 μ g/ml gelatin, 0.5 μ mol/L respectively detect with primer, wherein PCR damping fluid is the Tris-Cl damping fluid of pH value 8.3, each detection with primer is respectively and detects totally 6 of the upstream primer of 3 kinds of pathogenic agent and downstream primers, as shown in table 1:
The Auele Specific Primer of table 1 Eperythrozoon, Leptospira, three kinds of pathogenic agent of toxoplasma gondii
Test kit of the present invention is stored in-20 ℃, reduces multigelation as far as possible.
Another object of the present invention be to provide mentioned reagent box detect whether infect Eperythrozoon, Leptospira, toxoplasma gondii in blood in the application of single or multiple pathogenic agent.
The using method of test kit of the present invention is:
1.PCR reaction system preparation: reaction cumulative volume is 25 μ l, comprises PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked.
2. set up positive reference substance and negative control product; Positive reference substance is Eperythrozoon, Leptospira, bow-shaped worm dna sample mix liquid; Negative control product are aseptic deionized water.
2.PCR augmentation detection: the PCR reaction solution mixing is placed in to 96 hole PCR pipes, then puts into PCR instrument and carry out PCR reaction.Reaction conditions is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
3. detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × sample-loading buffer (Loading Buffer), with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.Positive control hole all have object band and negative control hole without the success of any band explanation PCR reaction detection.The electrophoretic band that tested sample aperture occurs and positive control hole respective strap is in the same size is judged to be the positive, on the contrary negative.
The useful gesture effect that the present invention compared with prior art has:
1) detection speed is fast: great advantage of the present invention is only to need a reaction can fast, accurately detect Eperythrozoon in test sample, Leptospira, three kinds of pathogenic agent DNA of toxoplasma gondii, also can be used for Molecule Epidemiology Investigation and the curative effect monitoring of Eperythrozoon, Leptospira, arch insect infection.Present method only can detect three kinds of parasitic DNA in 96 hole PCR pipes simultaneously, during in particular for the extensive detection of a large amount of samples, simple to operate, required sample dosage can greatly reduce, not only time saving and energy saving but also saved a large amount of reagent and consumptive material, improve working efficiency.
2) detection sensitivity is high, specificity good: by molecular biology software and pubmed database compare of analysis, select respectively three kinds of pathogen gene of high specificity, utilize oligo software to analyse and compare to primer, PCR reaction system is adjusted, thereby prepared the detection kit that susceptibility is high, specificity is good.This test kit can detect the DNA content of 0.1ng, and to other common disease substance no cross reaction.
3) easy and simple to handle: normal PCR time will add PCR Buffer, dNTP, primer, Taq enzyme, DNA step by step in operation, time-consuming, consumption power, error rate is high, easily pollute, and is not suitable for large batch of sample detection.This detection kit configures PCR reaction solution according to reaction conditions, only need add PCR reaction solution, Taq enzyme, DNA in use, greatly short operating time and consumptive material quantity used under the prerequisite that does not affect result, also reduced the probability polluting to a certain extent.
The present invention be based upon that a kind of susceptibility on molecular biology mechanism is high, high specificity, simple, fast detection method, can differentiate detection to the sample of single or multiple polyinfection red cell bodies, Leptospira, toxoplasma gondii simultaneously, greatly improve Detection accuracy, reduced false positive rate.
Accompanying drawing explanation
Fig. 1 is the standard multiplex PCR collection of illustrative plates of known red cell body, Leptospira, toxoplasma gondii, wherein M:Marker; Swimming lane 1: red cell body PCR amplified band (186bp); Swimming lane 2: Leptospira pcr amplification band (293bp); Swimming lane 3: PCR of toxoplasmas amplified band (407bp); Swimming lane 4: red cell body, Leptospira, toxoplasma gondii multiplex PCR amplified band; Swimming lane 5: negative control;
Fig. 2 is the multiplex PCR collection of illustrative plates of embodiment 1～9, wherein M:Marker; The pcr amplification band of swimming lane 1～9: embodiment 1～9; Swimming lane 10: positive control; 11: negative control.
Embodiment
For further analysis to the present invention below in conjunction with specific embodiment.
Embodiment positive reference substance used is Eperythrozoon, Leptospira, bow-shaped worm dna sample mix liquid below; Negative control product are aseptic deionized water.
Step (1). sample DNA extracts
Getting can hypochondriasis pig pig blood 0.5ml, extracts DNA by general genome DNA extracting reagent kit specification sheets, obtains DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is electrophoretic band in the 186bp place of swimming lane 1, illustrates in this DNA sample and contain red cell body; There is electrophoretic band in the 293bp place of swimming lane 1, illustrates in this DNA sample and contain Leptospira; There is not electrophoretic band in the 407bp place of swimming lane 1, illustrates in this DNA sample and do not contain toxoplasma gondii.Therefore this can infect red cell body, two kinds of pathogenic agent of Leptospira by hypochondriasis pig.
Step (1). sample DNA extracts
Get health pig pig blood 0.5ml, extract DNA by general genome DNA extracting reagent kit specification sheets, obtain DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is not electrophoretic band in the 186bp place of swimming lane 2, illustrates in this DNA sample and do not contain red cell body; There is not electrophoretic band in the 293bp place of swimming lane 2, illustrates in this DNA sample and do not contain Leptospira; There is not electrophoretic band in the 407bp place of swimming lane 2, illustrates in this DNA sample and do not contain toxoplasma gondii.Therefore this health pig has not infected red cell body, Leptospira, three kinds of pathogenic agent of toxoplasma gondii.
Step (1). sample DNA extracts
Getting can hypochondriasis ox ox blood 0.5ml, extracts DNA by general genome DNA extracting reagent kit specification sheets, obtains DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is electrophoretic band in the 186bp place of swimming lane 3, illustrates in this DNA sample and contain red cell body; There is electrophoretic band in the 293bp place of swimming lane 3, illustrates in this DNA sample and contain Leptospira; There is electrophoretic band in the 407bp place of swimming lane 3, illustrates in this DNA sample and contain toxoplasma gondii.Therefore this can hypochondriasis cattle infected red cell body, Leptospira, three kinds of pathogenic agent of toxoplasma gondii.
Step (1). sample DNA extracts
Get healthy ox ox blood 0.5ml, extract DNA by general genome DNA extracting reagent kit specification sheets, obtain DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is not electrophoretic band in the 186bp place of swimming lane 4, illustrates in this DNA sample and do not contain red cell body; There is not electrophoretic band in the 293bp place of swimming lane 4, illustrates in this DNA sample and do not contain Leptospira; There is not electrophoretic band in the 407bp place of swimming lane 4, illustrates in this DNA sample and do not contain toxoplasma gondii.Therefore this health ox has not infected red cell body, Leptospira, three kinds of pathogenic agent of toxoplasma gondii.
Step (1). sample DNA extracts
Getting can hypochondriasis chicken chicken blood 0.5ml, extracts DNA by general genome DNA extracting reagent kit specification sheets, obtains DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is electrophoretic band in the 186bp place of swimming lane 5, illustrates in this DNA sample and contain red cell body; There is not electrophoretic band in the 293bp place of swimming lane 5, illustrates in this DNA sample and do not contain Leptospira; There is electrophoretic band in the 407bp place of swimming lane 5, illustrates in this DNA sample and contain toxoplasma gondii.Therefore this can infect red cell body, two kinds of pathogenic agent of toxoplasma gondii by hypochondriasis chicken.
Step (1). sample DNA extracts
Get healthy chicken chicken blood 0.5ml, extract DNA by general genome DNA extracting reagent kit specification sheets, obtain DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is not electrophoretic band in the 186bp place of swimming lane 6, illustrates in this DNA sample and do not contain red cell body; There is not electrophoretic band in the 293bp place of swimming lane 6, illustrates in this DNA sample and do not contain Leptospira; There is not electrophoretic band in the 407bp place of swimming lane 6, illustrates in this DNA sample and do not contain toxoplasma gondii.Therefore this healthy chicken has not infected red cell body, Leptospira, three kinds of pathogenic agent of toxoplasma gondii.
Step (1). sample DNA extracts
Obtain ill patient's blood sample 0.5ml from hospital, extract DNA by general genome DNA extracting reagent kit specification sheets, obtain DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is not electrophoretic band in the 186bp place of swimming lane 7, illustrates in this DNA sample and do not contain red cell body; There is not electrophoretic band in the 293bp place of swimming lane 7, illustrates in this DNA sample and do not contain Leptospira; There is electrophoretic band in the 407bp place of swimming lane 7, illustrates in this DNA sample and contain toxoplasma gondii.Therefore this ill patient infection toxoplasmosis substance.
Step (1). sample DNA extracts
Obtain Healthy People blood sample 0.5ml from hospital,, extract DNA by general genome DNA extracting reagent kit specification sheets, obtain DNA sample to be checked.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000 r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is not electrophoretic band in the 186bp place of swimming lane 8, illustrates in this DNA sample and do not contain red cell body; There is not electrophoretic band in the 293bp place of swimming lane 8, illustrates in this DNA sample and do not contain Leptospira; There is not electrophoretic band in the 407bp place of swimming lane 8, illustrates in this DNA sample and do not contain toxoplasma gondii.Therefore this Healthy People has not infected red cell body, Leptospira, three kinds of pathogenic agent of toxoplasma gondii.
Step (1). sample DNA preparation
Extract respectively Eperythrozoon, Leptospira, bow-shaped worm dna by general genome DNA extracting reagent kit specification sheets, adjust to respectively 0.12ng/ μ l with aseptic deionized water, respectively get 20 μ l and mix, the contain Eperythrozoon, Leptospira, bow-shaped worm dna concentration that finally obtain are 0.04ng/ μ l.
Step (2) .PCR reaction system preparation: reaction cumulative volume is 25 μ l, PCR reaction solution is placed on ice and is melted, get successively PCR reaction solution 22.38 μ l, Taq archaeal dna polymerase 0.12 μ l, DNA sample 2.5 μ l to be checked mix in 96 hole PCR pipes, after mixing with pipettor piping and druming, insert the centrifugal 2s of whizzer 5000r/min.Set up positive reference substance and negative control product simultaneously.
Step (3) .PCR augmentation detection: the reaction tubes mixing is placed in to PCR instrument, reaction conditions is set, carry out PCR reaction.Reaction conditions is set is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 56.3 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 7min.
Step (4). detected result is judged: after pcr amplification reaction finishes, 2.5 μ l amplified productions are mixed with 0.2 μ l6 × Loading Buffer, with 1.5 ﹪ agarose gel electrophoresis detections, according to gel imaging system, imaging obtains corresponding electrophoretic band judged result.As shown in Figure 2, there is electrophoretic band in the 186bp place of swimming lane 9, illustrates in this DNA sample and contain red cell body; There is electrophoretic band in the 293bp place of swimming lane 9, illustrates in this DNA sample and contain Leptospira; There is electrophoretic band in the 407bp place of swimming lane 9, illustrates in this DNA sample and contain toxoplasma gondii.Therefore verified that this detection kit can detect the three kinds of pathogenic agent DNA of red cell body, Leptospira, toxoplasma gondii that contain 0.1ng simultaneously.
Above-described embodiment is not that the present invention is not limited only to above-described embodiment for restriction of the present invention, as long as meet requirement of the present invention, all belongs to protection scope of the present invention.
SEQUENCE LISTING
<110> Zhejiang University
Test kit and the application of Eperythrozoon, Leptospira, toxoplasma gondii in <120> rapid detection blood
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<212> DNA
<213> synthetic
<400> 6
atccgtatgc acaattcc 18
Claims (3)
1. a test kit for Eperythrozoon, Leptospira, toxoplasma gondii in rapid detection blood, is characterized in that this test kit is made up of detachable 96 hole PCR pipes, PCR reaction solution, Taq archaeal dna polymerase, standard substance, reference substance;
Described standard substance are three kinds of DNA samples that contain respectively Eperythrozoon, Leptospira, bow-shaped worm dna fragment;
Described reference substance is divided into positive reference substance and negative control product; Wherein positive reference substance is Eperythrozoon, Leptospira, bow-shaped worm dna sample mix liquid, and negative control product are aseptic deionized water;
Described PCR reaction solution comprises 10mmol/L PCR damping fluid, 0.5mmol/L dNTPs, 1.5mmol/L MgCl
2, 50mmol/L KCl, 100 μ g/ml gelatin, 0.5 μ mol/L respectively detect with primer, wherein PCR damping fluid is that pH value is 8.3 Tris-Cl damping fluid, detects with primer and is respectively and detects Eperythrozoon, Leptospira, 3 couples of Auele Specific Primer F1/R1, F2/R2 of toxoplasma gondii, F3/R3;
The Auele Specific Primer F1/R1 of described Eperythrozoon, length is 186bp:
Upstream primer F1:5 '-CTCCGCTCCTCCTTTTAT-3 ', the sequence as shown in SEQ ID NO:1;
Downstream primer R1:5 '-CAGGACTTAACACATCGGC-3 ', the sequence as shown in SEQ ID NO:2;
Described leptospiral Auele Specific Primer F2/R2, length is 293bp:
Upstream primer F2:5 '-ACCTAGGACAATTAGGAGG-3 ', the sequence as shown in SEQ ID NO:3;
Downstream primer R2:5 '-GTCGGACTCATAACCTACC-3 ', the sequence as shown in SEQ ID NO:4;
The Auele Specific Primer F3/R3 of described toxoplasma gondii, length is 407bp:
Upstream primer F3:5 '-CGAATGAAGAGTTGGCTGT-3 ', the sequence as shown in SEQ ID NO:5;
Downstream primer R3:5 '-ATCCGTATGCACAATTCC-3 ', the sequence as shown in SEQ ID NO:6.
In a kind of rapid detection blood as claimed in claim 1 the test kit of Eperythrozoon, Leptospira, toxoplasma gondii detect whether infect Eperythrozoon, Leptospira, toxoplasma gondii in blood in the application of single or multiple pathogenic agent.
3. the test kit of Eperythrozoon, Leptospira, toxoplasma gondii in a kind of rapid detection blood as claimed in claim 1, is characterized in that this test kit is stored in-20 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410034061.XA CN103789430B (en) | 2014-01-24 | 2014-01-24 | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410034061.XA CN103789430B (en) | 2014-01-24 | 2014-01-24 | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit |
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| CN103789430A true CN103789430A (en) | 2014-05-14 |
| CN103789430B CN103789430B (en) | 2015-05-20 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104862421A (en) * | 2014-07-11 | 2015-08-26 | 中华人民共和国济南出入境检验检疫局 | Complete set of products for identifying rabies, canine distemper, infectious hepatitis, canine parvovirus diseases, typhus billosus nostras and toxoplasmosis pathogeny |
| CN113186317A (en) * | 2021-05-31 | 2021-07-30 | 上海基灵生物科技有限公司 | Nucleic acid composition and kit for detecting zoonosis pathogen and application thereof |
| CN113493850A (en) * | 2021-08-19 | 2021-10-12 | 河北工程大学 | PCR primer probe group and kit for real-time fluorescent quantitative detection of toxoplasma gondii and eperythrozoon of pig and detection method thereof |
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| CN1850991A (en) * | 2006-02-27 | 2006-10-25 | 浙江大学 | Swine red cell body PCR detecting method |
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| CN1850991A (en) * | 2006-02-27 | 2006-10-25 | 浙江大学 | Swine red cell body PCR detecting method |
| CN101288774A (en) * | 2007-11-08 | 2008-10-22 | 上海交通大学 | Construction method of mice infection model of Suis Eperythrozoonsis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104862421A (en) * | 2014-07-11 | 2015-08-26 | 中华人民共和国济南出入境检验检疫局 | Complete set of products for identifying rabies, canine distemper, infectious hepatitis, canine parvovirus diseases, typhus billosus nostras and toxoplasmosis pathogeny |
| CN113186317A (en) * | 2021-05-31 | 2021-07-30 | 上海基灵生物科技有限公司 | Nucleic acid composition and kit for detecting zoonosis pathogen and application thereof |
| CN113493850A (en) * | 2021-08-19 | 2021-10-12 | 河北工程大学 | PCR primer probe group and kit for real-time fluorescent quantitative detection of toxoplasma gondii and eperythrozoon of pig and detection method thereof |
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| Publication number | Publication date |
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| CN103789430B (en) | 2015-05-20 |
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