CN1786193A - Endogenous reference used for detecting human body pathogen and its application - Google Patents
Endogenous reference used for detecting human body pathogen and its application Download PDFInfo
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- CN1786193A CN1786193A CNA2004100893718A CN200410089371A CN1786193A CN 1786193 A CN1786193 A CN 1786193A CN A2004100893718 A CNA2004100893718 A CN A2004100893718A CN 200410089371 A CN200410089371 A CN 200410089371A CN 1786193 A CN1786193 A CN 1786193A
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Abstract
The invention discloses an examination human body pathogen endogenous reference and its application. And it offers biological sample identity system amplification human body pathogen DNA and body endogenous reference gene DNA method and its corresponding nucleic acid examination kit. The endogenous reference can use as monitoring reference in overall process to effectively reduce the examination false negative probability.
Description
Technical field
The present invention relates to the detection method of nucleic acid, relate to the endogenous reference and the application thereof that are used for a kind of human body pathogenic agent particularly.The method of increase in same system from biological sample human body pathogen DNA and human body endogenous crt gene DNA also is provided, and the corresponding nucleic acids detection kit.
Background technology
It is a leap that virus detects from biochemical method to the nucleic acid method.Since the PCR method invention, detection of nucleic acids has obtained the development of advancing by leaps and bounds, and combines with other technology various detection techniques to occur.
The development of PCR detection technique is roughly as follows: begin to send by being attached to EB on the nucleic acid double chain that fluorescence detects with agarose gel electrophoresis method under the irradiation of ultraviolet, shortcoming is that open system easily pollutes, and EB and ultraviolet are harmful.Fluorescent probe technique afterwards is introduced among the PCR, and principle is at first to excite to be attached to the fluorophor that the fluorescently-labeled probe on the target sequence sends, and detects that the fluorescence send detects again.Fluorescent PCR has end-point method and real time method again, the former is first level method, characteristics are quantitatively inaccurate, real time method is wanted accurately relatively, real time method is a main flow with the Taqman technology, what the domestic overwhelming majority used is the Taqman technology, and this is by Roche Molecular System Inc (Roche Holding Ag) invention, and has patent at western developed country.
PCR is owing to have the specificity and the susceptibility of height, develops very fastly, becomes the development trend of visiting.In the development in several years, round pcr is progressively perfect, but the false negative problem is the difficult problem of puzzlement visiting circle always.Add confidential reference items template part ground and solve this difficult problem, but can not detect the whole process that comprises in being extracted in, and the endogenous confidential reference items can be avoided above shortcoming.
Yet up to the present this area still lacks gratifying endogenous confidential reference items.Therefore this area presses for effectively endogenous confidential reference items of exploitation, so that further reduce false negative rate.
Summary of the invention
Purpose of the present invention is exactly a kind of effective endogenous confidential reference items, can reduce the false negative rate with PCR method human body pathogenic agent DNA effectively.
In a first aspect of the present invention, a kind of human body endogenous dna contrast is provided, it has the nucleotide sequence shown in the SEQ IDNO:1.
In a second aspect of the present invention, the purposes of the above-mentioned human body endogenous dna of the present invention contrast is provided, it is used as the object of reference of the DNA when increasing human body pathogen in same amplification system from biological sample.
In another preference, in described amplification system, contain be selected from the amplification this human body endogenous dna contrast primer to with amplified production bonded probe.
In another preference, described primer to probe be selected from following each the group:
Group (a):
Upstream primer 1 GGCTGAGAGGGCGTAGGAATT (SEQ ID NO:2)
Downstream primer 1 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:3)
Probe 1 GCGAGGATGAAACCGATATCG (SEQ ID NO:4)
Group (b):
Upstream primer 2 TCGGCTGAGAGGGCGTAGGAAT (SEQ ID NO:5)
Downstream primer 2 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:6)
Probe 2 GGCGAGGATGAAACCGATATCG (SEQ ID NO:7)
Group (c):
Upstream primer 3 ATCGGCTGAGAGGGCGTAGGAA (SEQ ID NO:8)
Downstream primer 3 TGATACGCCCGAGCAGATGCCA (SEQ ID NO:9)
Probe 3 GGCGAGGATGAAACCGATATCG (SEQ ID NO:10)
Group (d):
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 CATGCTAAGGCGAGGATGAAAC (SEQ ID NO:13).
In another preference, described human body pathogen comprises bacterium, chlamydozoan, mycoplasma or dna virus.
In another preference, wherein said biological sample is selected from human blood, serum, blood plasma, saliva, cerebrospinal fluid, somatocyte.
In a third aspect of the present invention, a kind of method that is used for vitro detection pathogenic agent DNA is provided, described method comprises the following steps:
(a) with the human body pathogen DNA that derives from described sample and human body endogenous crt gene DNA as template, with first primer of specific amplification human body pathogen DNA to second primer of specific amplification human body endogenous crt gene DNA to as Oligonucleolide primers, carry out nucleic acid amplification reaction, thereby produce first specific amplification products of human body pathogen DNA and second specific amplification products of human body endogenous crt gene DNA;
(b) whether the existence of described first specific amplification products of detection and second specific amplification products;
Wherein, the existence of described two specific specificity amplified productions just shows that human body pathogen DNA and human body endogenous crt gene DNA are present in the described sample; Second specific amplification products that does not have human body endogenous crt gene DNA just represents that detected result is a false negative.
More preferably, described amplification is selected from down group: polymerase chain reaction (PCR), ligase chain reaction (LCR), chain substitute amplification (SDA) or rolling circle amplification (RCA).
More preferably, described detection is to be undertaken by being selected from following method: gelose gel electrophoresis, acrylamide gel electrophoresis method, fluorescent probe detection method, molecular beacon fluorescence detection, radio isotope detection method and chemiluminescence detecting method.
In a fourth aspect of the present invention, a kind of detection kit is provided, it is right that it contains the primer of the above-mentioned human body endogenous dna contrast of specific amplification the present invention, described primer length is a 15-35 Nucleotide, and the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1, and the length of amplified production is 100-1000bp.
Preferably, described test kit also contains and amplified production bonded probe, and the length of described probe is 20-50bp, and the sequence of this probe is identical or complementary with the sequence shown in the SEQ ID NO:1.
In another preference, the primer described in the described test kit to probe be selected from following each the group:
Group (a):
Upstream primer 1 GGCTGAGAGGGCGTAGGAATT (SEQ ID NO:2)
Downstream primer 1 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:3)
Probe 1 GCGAGGATGAAACCGATATCG (SEQ ID NO:4)
Group (b):
Upstream primer 2 TCGGCTGAGAGGGCGTAGGAAT (SEQ ID NO:5)
Downstream primer 2 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:6)
Probe 2 GGCGAGGATGAAACCGATATCG (SEQ ID NO:7)
Group (c):
Upstream primer 3 ATCGGCTGAGAGGGCGTAGGAA (SEQ ID NO:8)
Downstream primer 3 TGATACGCCCGAGCAGATGCCA (SEQ ID NO:9)
Probe 3 GGCGAGGATGAAACCGATATCG (SEQ ID NO:10)
Group (d):
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 CATGCTAAGGCGAGGATGAAAC (SEQ ID NO:13).
In another preference, described test kit also contain the Oligonucleolide primers that detects hepatitis B virus DNA to and probe, described primer is to being selected from down group with probe:
Group (e):
Upstream primer 5 TGCCAAGTGTTTGCTGA (SEQ ID NO:14)
Downstream primer 5 AAGAAGGGGACGGTAGA (SEQ ID NO:15)
Probe 5 ACGCAACCCCCACTGGCTGGGG (SEQ ID NO:16)
Group (f):
Upstream primer 6 TAGGACCCCTGCTCGTG (SEQ ID NO:17)
Downstream primer 6 ATGGGATGGGAATACAAG (SEQ ID NO:18)
Probe 8 AGGCGGGGTTTTTCTTGTTGA (SEQ ID NO:19)
Group (g):
Upstream primer 7 GCACTTGTATTCCCATCC (SEQ ID NO:20)
Downstream primer 7 AAAGCCCTACGAACCAC (SEQ ID NO:21)
Probe 7 ATCCCATCATCCTGGGCTTTCG (SEQ ID NO:22)
Group (h):
Upstream primer 8 TGCCAAGTGTTTGCTGA (SEQ ID NO:23)
Downstream primer 8 AGAAGGGGACGGTAGATC (SEQ ID NO:24)
Probe 8 CCACTGGCTGGGGCTTGGCCATT (SEQ ID NO:25);
Perhaps also contain the Oligonucleolide primers that detects ureaplasma urealyticum DNA to and probe, described primer is to being selected from down group with probe:
Upstream primer: 5 ' TGGTT TGTCC GCATC ATGGT-3 ' (SEQ ID NO:27)
Downstream primer; 5 ' AGTCC ATGAT GAAGA ACCAA-3 ' (SEQ ID NO:28)
Probe: FAM-TGACG GGCTA TATGT GGATC-TAMRA (SEQ ID NO:29);
Perhaps also contain the Oligonucleolide primers that detects gonococcus DNA to and probe, described primer is to being selected from down group with probe:
Upstream primer: 5 '-GGTTG GAAGC CGTGA AGCTG-3 ' (SEQ ID NO:30)
Downstream primer; 5 '-GGTTC AGTGA CAGTG AGCTA-3 ' (SEQ ID NO:31)
Probe: FAM-TTGAG CGTGT GAGAT AGTGC AG-TAMRA (SEQ ID NO:32);
Perhaps also contain the Oligonucleolide primers that detects the former DNA of trachoma clothing to and probe, described primer is to being selected from down group with probe:
Upstream primer: 5 '-TGATG GATCG ATGAC AGTCG-3 ' (SEQ ID NO:33)
Downstream primer; 5 '-GTGTA AGTCA GTGAT AGCGT-3 ' (SEQ ID NO:34)
Probe: FAM-GTGTA TGACT GATCG CGTGA GT-TAMRA (SEQ ID NO:35).
Embodiment
Extensive studies finds that a kind of sequence is particularly suitable as internal control gene to the inventor through going deep into, and it can keep the stable of content preferably in the method for extracting of routine.Therefore can be used as the object of reference of PCR process, extract or extractive process, thereby can be used as the object of reference of monitoring whole process, therefore can reduce probability of false negative effectively but also can be used as monitoring.Finished the present invention on this basis.
As used herein, term " nucleic acid " or " polynucleotide " refer to the purine-containing of random length and contain the polymkeric substance of pyrimidine, can be the strand and the duplex molecule of poly deoxynucleosides, also comprise the nucleic acid that contains base modification.
As used herein, term " primer " is meant the oligonucleotide between a kind of about 10 to 60 length of nucleotides, preferably about 12-50 Nucleotide, more preferably about 15-40 Nucleotide, about best 18-25 length of nucleotides.Primer can be incorporated into template, and under such as the catalysis of archaeal dna polymerase the polymerization complementary strand, form amplified production.
As used herein, term " extraction " or " extracting " refer to from its primal environment according to isolated one or more compositions of certain working method, especially isolate the nucleic acid molecule as amplification template.
As used herein, term " endogenous " gene refers to from the genomic DNA of biological sample.
Nucleic acid molecule as used herein, that term " endogenous reference " or " internal control gene " refer to have sequence shown in the SEQ ID NO:1.Should be understood that this term comprises that also the length that is derived from SEQ ID NO:1 is at least 100bp, 200bp at least preferably, 400bp at least more preferably, the nucleic acid fragment of 500bp at least best.
As used herein, term " amplification " refers to a kind of repetitive process, can replicating nucleic acid by this process.The appropriate method that is used to increase includes but not limited to: polymerase chain reaction (PCR), ligase chain reaction (LCR), chain substitutes amplification (SDA), rolling circle amplification (RCA).
According to the present invention, provide basis for estimation to human body pathogen DNA cloning detected result to the augmentation detection result of human body endogenous crt gene DNA.
Can be used for amplified reaction of the present invention is not particularly limited.Representational amplified reaction includes, but is not limited to: polymerase chain reaction (PCR), ligase chain reaction (LCR), chain substitutes amplification (SDA), rolling circle amplification (RCA).Preferential polymerase chain reaction (PCR).
Detection reaction is used with the corresponding any means of amplification and is carried out.Preferred detection method comprises (but being not limited to): gelose gel electrophoresis, acrylamide gel electrophoresis method, fluorescent probe detection method, molecular beacon fluorescence detection, radio isotope detection method and chemiluminescence detecting method.The preferred detection method of mating with PCR is the Taqman fluorescence detection method.
Taqman fluorescent probe detection method is to know in this area, and it utilizes the Tacman probe, detects simultaneously in same system and increases, and calculate the concentration of primary template according to certain algorithm by the detection to the fluorescent value of different wave length.
The pathogenic agent that available the inventive method detects is not particularly limited, and representational example comprises (but being not limited to): HBV, ureaplasma urealyticum UU, gonococcus NG, the former CT of trachoma clothing etc.
In a preference, detected pathogenic agent is a hepatitis B virus.As used herein, term hepatitis B virus (HBV) refers to the virus in the Hepacivirus genus.Can comprise any HBV serotype by the HBV isolate that the present invention detects.When being used to detect HBV, can comprise positive object of reference of HBV or the quantitatively positive product of HBV in described method or described test kit.Positive object of reference of HBV or the quantitatively positive product of HBV refer to a kind of nucleotide sequence that is cloned into plasmid vector, and this sequence is identical or complementary with the sequence in one section zone of HBV whole genome sequence.The sequence of the quantitative object of reference of a kind of preferred HBV is shown in SEQ ID NO:26.
According to the present invention, biological sample by any conventional mode available from the patient.Suitable biological sample includes but not limited to: blood, serum, blood plasma, urine, saliva, milk, swab, seminal fluid or cerebrospinal fluid, and composition thereof.
The human body pathogen DNA that is used for increasing can separate from above-mentioned biological sample with human body endogenous crt gene DNA.Can use method well-known in the art or commercially available reagent, extracting or separation are as the nucleic acid molecule of amplification template from sample, and separation method should guarantee that separated product contains human body pathogen DNA and human body endogenous crt gene DNA.
Use then and derive from the primer of human body pathogen DNA and the primer of human body endogenous crt gene DNA carries out amplified reaction to the biological sample isolate in same system.Primer design is conventional to those skilled in the art.The primer of the primer of the human body pathogen DNA that selects for use and human body endogenous crt gene DNA can be according to well-known design of primers principle, and can and also only with the sequence of each self-corresponding human body pathogen DNA specific region and the sequence hybridization of human body endogenous crt gene DNA specific region.Preferably, the primer of the primer of human body pathogen DNA and human body endogenous crt gene DNA can be under identical conditions and same system in target sequence is separately increased.
Being used for primer of the present invention and probe can prepare by ordinary method.For example, use phosphoramidite solid Zhi Chifa or other known method can be with the chemical mode synthetic DNAs.Can also modify described nucleic acid by many modes well known in the art.The limiting examples that this class is modified comprises and methylating etc.Nucleic acid can contain one or more other part with the covalent connection, for example protein, intercalator, sequestrant and alkylating agents.Nucleotide sequence of the present invention can be modified in direct or indirect mode by the marker that can provide detectable signal.Typical marker comprises radio isotope, fluorescence molecule, vitamin H.
Endogenous of the present invention is with reference to being particularly suitable in same system DNA amplification.As used herein, term " is applicable to the condition of same system DNA amplification " refers to use Oligonucleolide primers DNA amplification synthetic condition, comprises temperature and time of the concentration content of volume, composition, each composition of amplification system and ratio, amplification etc.
On the other hand, the invention provides the method for from biological sample, in same system, increase human body pathogen DNA and human body endogenous crt gene DNA.Derive from human body pathogen DNA and human body endogenous crt gene DNA and corresponding Oligonucleolide primers of the present invention by making, under the specificity condition, increase, whether the amplified production that detects pathogenic agent amplified production and endogenous object of reference then exists, if wherein there is the amplified production of endogenous object of reference in the product after the amplification, show that then test-results is not a false negative: the if there is no amplified production of endogenous object of reference in the product after the amplification shows that then test-results is a false negative.
If, can obtain detected result more accurately in conjunction with positive control (being used to get rid of false positive).
Particularly, the detection of amplified production DNA in a kind of preferred biological sample can be carried out through the following steps: the DNA that uses the DNA that contains in the described sample or derive from described sample makes template, use increasing as amplimer of containing among the present invention with human body pathogen DNA and human body endogenous crt gene dna sequence dna complementary oligonucleotide, thereby produce two species specific amplified productions, simultaneously or subsequently two specific specificity amplified productions are detected.Detected result to human body pathogen DNA cloning product shows whether there is this human body pathogen in the described sample, and the detected result of human body endogenous crt gene DNA cloning product is shown whether this amplification system amplification procedure is effective.
In another aspect of this invention, provide that to contain with endogenous object of reference of the present invention be the detection kit of false negative contrast.This test kit is used for test kit and the human body endogenous crt gene DNA of detection of biological sample human body pathogen DNA.Typically, this test kit comprises the primer of amplification pathogenic agent primer and amplification human body endogenous crt gene DNA.Preferably, described test kit also can comprise be used to extract, reagent and specification sheets that amplification and product detect usefulness.
Method of the present invention and test kit can be used for the blood of diagnosis, detection result of treatment and the screening DNA pathogenic infection of patient DNA pathogenic agent.
Major advantage of the present invention is that endogenous reference of the present invention can be used as the object of reference of the whole process of monitoring from the sample drawn to the pcr amplification, thereby can reduce the probability of false negative of detection effectively.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment
Universal method
1, specimen preparation:
Use multiple star dna virus to extract the ordinary method described in reagent (available from the multiple star limited-liability company in Shanghai, Shanghai, China) or " molecular cloning ", from serum specimen, prepare DNA.This method adopts the hepatitis B virus particles in the concentrated serum sample of polyoxyethylene glycol (PEG) precipitator method, alkaline lysis retrovirus particle, and Chelex absorption impurity obtains DNA.
2, pcr amplification
On the automatic fluorescent PCR instrument of Rotogene, increase, reaction system is made up of following ingredients: 16.7mmol/L Tris-HCl (pH8.3), 83.3mmol/L KCl, dATP, dGTP, dCTP, dUTP0.333mmol/L, each 0.50 μ mol/L of primer, probe 2pmol/ul, MgCl2 35mmol/L, Taq enzyme 3U/test. is earlier 50 ℃ of reactions 1 minute, and 94 ℃ are incubated 2 minutes then, again by 94 ℃ 2 seconds → 55 ℃ 72 ℃ of circulations in 10 seconds in 15 seconds 40 times.
The Oligonucleolide primers and the probe of human body endogenous crt gene dna sequence dna of being used to increase is selected from table 1.
The primer and the probe of the reference of table 1 amplification endogenous
Group (a):
Upstream primer 1 GGCTGAGAGGGCGTAGGAATT (SEQ ID NO:2)
Downstream primer 1 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:3)
Probe 1 GCGAGGATGAAACCGATATCG (SEQ ID NO:4)
Group (b):
Upstream primer 2 TCGGCTGAGAGGGCGTAGGAAT (SEQ ID NO:5)
Downstream primer 2 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:6)
Probe 2 GGCGAGGATGAAACCGATATCG (SEQ ID NO:7)
Group (c):
Upstream primer 3 ATCGGCTGAGAGGGCGTAGGAA (SEQ ID NO:8)
Downstream primer 3 TGATACGCCCGAGCAGATGCCA (SEQ ID NO:9)
Probe 3 GGCGAGGATGAAACCGATATCG (SEQ ID NO:10)
Group (d):
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 CATGCTAAGGCGAGGATGAAAC (SEQ ID NO:13).
Oligonucleolide primers and the probe of human body hepatitis B virus (HBV) DNA of being used to increase is selected from table 2.
Primer and the probe of table 2 amplification HBV
Group (e):
Upstream primer 5 TGCCAAGTGTTTGCTGA (SEQ ID NO:14)
Downstream primer 5 AAGAAGGGGACGGTAGA (SEQ ID NO:15)
Probe 5 ACGCAACCCCCACTGGCTGGGG (SEQ ID NO:16)
Group (f):
Upstream primer 6 TAGGACCCCTGCTCGTG (SEQ ID NO:17)
Downstream primer 6 ATGGGATGGGAATACAAG (SEQ ID NO:18)
Probe 6 AGGCGGGGTTTTTCTTGTTGA (SEQ ID NO:19)
Group (g):
Upstream primer 7 GCACTTGTATTCCCATCC (SEQ ID NO:20)
Downstream primer 7 AAAGCCCTACGAACCAC (SEQ ID NO:21)
Probe 7 ATCCCATCATCCTGGGCTTTCG (SEQ ID NO:22)
Group (h):
Upstream primer 8 TGCCAAGTGTTTGCTGA (SEQ ID NO:23)
Downstream primer 8 AGAAGGGGACGGTAGATC (SEQ ID NO:24)
Probe 8 CCACTGGCTGGGGCTTGGCCATT (SEQ ID NO:25).
The primer and the probe that are used for other pathogenic agent are listed in each embodiment.
3, amplified production detects
Adopt the Taqman probe, with pathogenic agent DNA specificity bonded probe mark FAM-(490nm), with human body endogenous crt gene DNA specificity bonded probe mark TET (530nm), each round-robin that fluoroscopic examination occurs in pcr amplification extends the stage. when carrying out quantitative analysis, select fluoroscopic examination model F 1/F2, under the Proportional account form, the baseline thresholding is transferred to 0.05 place, utilize the automatic fluorescent PCR instrument of Rotogene software kit to calculate the result of unknown sample.
Embodiment 1
Whole blood, serum and plasma sample detected result are relatively
Get the serum in same source, blood plasma, positive control, negative control are respectively got 50 μ l in whole blood sample, the test kit, add 50 μ l nucleic acid extraction liquid A respectively, 10 seconds kinds of vibration mixing, 12, centrifugal 10 minutes of 000rpm abandons supernatant; Add 50 μ l nucleic acid extraction liquid B (abundant mixing when nucleic acid extraction liquid B uses adds particle and liquid in the precipitation together, and wherein grain amount should be in 15%) to precipitation respectively, vibration 10 seconds of mixing, 100 ℃ of boiling water baths 10 minutes, 12, centrifugal 2 minutes of 000rpm.
Pcr amplification and fluorescence detection method as above, system only adds human body endogenous crt gene dna primer and probe.The result is as follows:
| The sample source | The CT value | Quantitative result (copies/ml) |
| Serum | 21.74 | 5.370×10 6 |
| The same | 21.73 | 5.373×10 6 |
| Blood plasma | 19.61 | 4.699×10 7 |
| The same | 19.62 | 4.697×10 7 |
| Whole blood | 17.85 | 2.570×10 9 |
| The same | 17.87 | 2.573×10 9 |
The result shows that in same extracting method, three kinds of samples contain the concentration of human body endogenous crt gene DNA: whole blood＞blood plasma＞serum.
Embodiment 2
Different magnesium ion concentrations influence the result in the system
With embodiment 1, magnesium ion concentration is pressed in the amplification system: 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM add respectively in the reaction system sample with the serum extracting method, and the same result is as follows for amplification program:
| Sample | The CT value | |||||
| 1.5mM | 2.0mM | 2.5mM | 3.0mM | 3.5mM | 4.0mM | |
| 156×10 7 | 22.63 | 21.83 | 21.35 | 21.21 | 20.95 | 20.95 |
| 156×10 6 | 26.59 | 25.24 | 24.86 | 24.60 | 24.42 | 24.42 |
| 156×10 5 | 29.36 | 29.08 | 28.48 | 28.32 | 28.32 | 28.32 |
| 156×10 4 | 33.85 | 32.39 | 31.90 | 31.03 | 31.81 | 31.81 |
| 156×10 3 | 36.75 | 35.92 | 35.48 | 35.32 | 35.03 | 35.03 |
| 156×10 2 | 39.77 | 37.56 | 36.75 | 36.47 | 36.63 | 35.93 |
The above results shows that the magnesium ion concentration of 2.0-10.0mM all can use, and wherein the magnesium ion concentration of 3.5mM is the most suitable.
Embodiment 3
Detect hepatitis B virus
Adopt the hepatitis B virus particles in the concentrated serum sample of polyoxyethylene glycol (PEG) precipitator method, alkaline lysis retrovirus particle, Chelex absorption impurity obtains DNA.
Extract the back sample and add reaction system, the system composition sees before, and contains HBV primer and probe in the system, confidential reference items primer and probe.
HBV primer and probe sequence are:
Upstream primer 5 TGCCAAGTGTTTGCTGA (SEQ ID NO:14)
Downstream primer 5 AAGAAGGGGACGGTAGA (SEQ ID NO:15)
Probe FAM-5 ACGCAACCCCCACTGGCTGGGG-TAMRA (SEQ ID NO:16)
Confidential reference items primer and probe sequence are:
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 FAM-CATGCTAAGGCGAGGATGAAAC-TAMRA (SEQ ID NO:13)
Augmentation detection ROTOGENE, earlier 50 ℃ of reactions 1 minute, 94 ℃ are incubated 2 minutes then, again by 94 ℃ 2 seconds → 55 ℃ 72 ℃ of circulations in 10 seconds in 15 seconds 40 times.Each round-robin that fluoroscopic examination occurs in pcr amplification extends the stage. when carrying out quantitative analysis, select fluoroscopic examination model F 1/F2, under the Proportional account form, the baseline thresholding is transferred to 0.05 place, utilize the automatic fluorescent PCR instrument of Rotogene software kit to calculate the result of unknown sample.
The result is as follows:
| The sample title | Known HBV concentration | CH1 (HBV) CT value | CH2 (IC) CT value |
| HBV quantitative 1 | 10 6 | 21.44 | / |
| HBV quantitative 2 | 10 5 | 24.46 | / |
| HBV quantitative 3 | 10 4 | 29.05 | / |
| Negative serum | 0 | / | 23.65 |
| HBV positive serum 1 | 10 7 | 20.66 | 24.03 |
| HBV positive serum 2 | 10 6 | 21.03 | 22.12 |
| HBV positive serum 3 | 10 5 | 24.82 | 23.76 |
| HBV positive serum 4 | 10 3 | 35.85 | 22.40 |
| Water | 0 | / | / |
The result shows, the detected result of HBV is consistent with expection, and the content of endogenous object of reference in each sample keeps constant basically, thereby is good endogenous object of reference.
Embodiment 4
Detect UU
Adopt the genitourinary tract swab sample, acquisition method is: autoclaved special cotton swab, stretch on male urethra mouth or the women's uterine neck mouth 1-2 centimetre, rotate a circle, take out after stopping about 10 seconds, the cotton swab head is put into the freezing preservation pipe of the sample that fills 1 milliliter of stroke-physiological saline solution, cut off swab from neck, make the swab head immerse in the salt solution rinsing repeatedly.Obtain separated DNA in cracking with separating then.Multiple star test kit is seen in concrete operations, extracts the back sample and adds reaction system, and the system composition sees before, and contains UU primer and probe in the system, confidential reference items primer and probe.
UU primer probe sequence is:
Upstream primer: 5 '-TGGTT TGTCC GCATC ATGGT-3 ' (SEQ ID NO:27)
Downstream primer; 5 '-AGTCC ATGAT GAAGA ACCAA-3 ' (SEQ ID NO:28)
Probe: FAM-TGACG GGCTA TATGT GGATC-TAMRA (SEQ ID NO:29).
Confidential reference items primer and probe sequence are:
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 FAM-CATGCTAAGGCGAGGATGAAAC-TAMRA (SEQ ID NO:13)
Augmentation detection ROTOGENE, earlier 50 ℃ of reactions 1 minute, 94 ℃ are incubated 2 minutes then, again by 94 ℃ 2 seconds → 55 ℃ 72 ℃ of circulations in 10 seconds in 15 seconds 40 times.Each round-robin that fluoroscopic examination occurs in pcr amplification extends the stage. when carrying out quantitative analysis, select fluoroscopic examination model F 1/F2, under the Proportional account form, the baseline thresholding is transferred to 0.05 place, utilize the automatic fluorescent PCR instrument of Rotogene software kit to calculate the result of unknown sample.
The result is as follows:
| The sample title | Known UU concentration | CH1 (UU) CT value | CH2 (IC) CT value |
| UU quantitative 1 | 10 6 | 21.33 | / |
| UU quantitative 2 | 10 5 | 24.64 | / |
| UU quantitative 3 | 10 4 | 29.50 | / |
| Negative serum | 0 | / | 23.55 |
| UU positive sample 1 | 10 7 | 20.45 | 24.30 |
| UU positive sample 2 | 10 6 | 21.34 | 22.21 |
| UU positive sample 3 | 10 5 | 24.87 | 23.78 |
| UU positive sample 4 | 10 3 | 35.55 | 22.04 |
| Water | 0 | / | / |
The result shows, the detected result of UU is consistent with expection, and the content of endogenous object of reference in each sample keeps constant basically, thereby is good endogenous object of reference.
Embodiment 5
Detect NG
Adopt the genitourinary tract swab sample, acquisition method is: autoclaved special cotton swab, stretch on male urethra mouth or the women's uterine neck mouth 1-2 centimetre, rotate a circle, take out after stopping about 10 seconds, the cotton swab head is put into the freezing preservation pipe of the sample that fills 1 milliliter of stroke-physiological saline solution, cut off swab from neck, make the swab head immerse in the salt solution rinsing repeatedly.Obtain separated DNA in cracking with separating then.Multiple star test kit is seen in concrete operations, extracts the back sample and adds reaction system, and the system composition sees before, and contains NG primer and probe in the system, confidential reference items primer and probe.
NG primer and probe sequence are:
Upstream primer: 5 '-GGTTG GAAGC CGTGA AGCTG-3 ' (SEQ ID NO:30)
Downstream primer; 5 '-GGTTC AGTGA CAGTG AGCTA-3 ' (SEQ ID NO:31)
Probe: FAM-TTGAG CGTGT GAGAT AGTGC AG-TAMRA (SEQ ID NO:32).
The primer of confidential reference items and probe sequence are:
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 FAM-CATGCTAAGGCGAGGATGAAAC-TAMRA (SEQ ID NO:13)
Augmentation detection ROTOGENE, earlier 50 ℃ of reactions 1 minute, 94 ℃ are incubated 2 minutes then, again by 94 ℃ 2 seconds → 55 ℃ 72 ℃ of circulations in 10 seconds in 15 seconds 40 times.Each round-robin that fluoroscopic examination occurs in pcr amplification extends the stage. when carrying out quantitative analysis, select fluoroscopic examination model F 1/F2, under the Proportional account form, the baseline thresholding is transferred to 0.05 place, utilize the automatic fluorescent PCR instrument of Rotogene software kit to calculate the result of unknown sample.
The result is as follows:
| The sample title | Known NG concentration | CH1 (NG) CT value | CH2 (IC) CT value |
| NG quantitative 1 | 10 6 | 21.33 | / |
| NG quantitative 2 | 10 5 | 24.35 | / |
| NG quantitative 3 | 10 4 | 29.04 | / |
| Negative serum | 0 | / | 23.65 |
| NG positive serum 1 | 10 7 | 20.64 | 24.03 |
| NG positive serum 2 | 10 6 | 21.06 | 22.12 |
| NG positive serum 3 | 10 5 | 24.86 | 23.76 |
| NG positive serum 4 | 10 3 | 35.78 | 22.40 |
| NG positive serum 5 | 10 3 | 39.70 | 23.12 |
| Water | 0 | / | / |
The result shows, the detected result of NG is consistent with expection, and the content of endogenous object of reference in each sample keeps constant basically, thereby is good endogenous object of reference.
Embodiment 6
Detect CT
Adopt the genitourinary tract swab sample, acquisition method is: autoclaved special cotton swab, stretch on male urethra mouth or the women's uterine neck mouth 1-2 centimetre, rotate a circle, take out after stopping about 10 seconds, the cotton swab head is put into the freezing preservation pipe of the sample that fills 1 milliliter of stroke-physiological saline solution, cut off swab from neck, make the swab head immerse in the salt solution rinsing repeatedly.Obtain separated DNA in cracking with separating then.Multiple star test kit is seen in concrete operations, extracts the back sample and adds reaction system, and the system composition sees before, and contains CT primer and probe in the system, confidential reference items primer and probe.
CT primer probe sequence is:
Upstream primer: 5 '-TGATG GATCG ATGAC AGTCG-3 ' (SEQ ID NO:33)
Downstream primer; 5 '-GTGTA AGTCA GTGAT AGCGT-3 ' (SEQ ID NO:34)
Probe: FAM-GTGTA TGACT GATCG CGTGA GT-TAMRA (SEQ ID NO:35)
The primer of confidential reference items and probe:
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 FAM-CATGCTAAGGCGAGGATGAAAC-TAMRA (SEQ ID NO:13)
Augmentation detection ROTOGENE, earlier 50 ℃ of reactions 1 minute, 94 ℃ are incubated 2 minutes then, again by 94 ℃ 2 seconds → 55 ℃ 72 ℃ of circulations in 10 seconds in 15 seconds 40 times.Each round-robin that fluoroscopic examination occurs in pcr amplification extends the stage. when carrying out quantitative analysis, select fluoroscopic examination model F 1/F2, under the Proportional account form, the baseline thresholding is transferred to 0.05 place, utilize the automatic fluorescent PCR instrument of Rotogene software kit to calculate the result of unknown sample.
The result is as follows:
| The sample title | Known CT concentration | CH1 (CT) CT value | CH2 (IC) CT value |
| CT quantitative 1 | 10 6 | 21.24 | / |
| CT quantitative 2 | 10 5 | 24.37 | / |
| CT quantitative 3 | 10 4 | 29.32 | / |
| Negative serum | 0 | / | 23.12 |
| CT positive serum 1 | 10 7 | 20.23 | 24.56 |
| CT positive serum 2 | 10 6 | 22.45 | 22.34 |
| CT positive serum 3 | 10 5 | 24.98 | 23.13 |
| CT positive serum 4 | 10 4 | 3154 | 22.20 |
| Water | 0 | / | / |
The result shows, the detected result of CT is consistent with expection, and the content of endogenous object of reference in each sample keeps constant basically, thereby is good endogenous object of reference.
Embodiment 7
Detect hepatitis B virus
Repeat embodiment 3, difference only is to replace confidential reference items upstream primer 4, downstream primer 4, the probe 4 of (d) group respectively with (a) in the table 1 group, (b) group or the primer of (c) organizing and probe.
The result is similar to embodiment 3.The detected result of HBV is consistent with expection, and the content of endogenous object of reference in each sample keeps constant basically, thereby is good endogenous object of reference.
Embodiment 8
Detect hepatitis B virus
Repeat embodiment 3, difference only is to replace HBV upstream primer 5, downstream primer 5, the probe 5 of (e) group respectively with (f) in the table 2 group, (g) group or the primer of (h) organizing and probe.
The result is similar to embodiment 3.The detected result of HBV with the expection consistent, show this endogenous object of reference can with different HBV primer and probe combinations.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Fuxing Medical Science-Technology Development Co., Ltd., Shanghai
＜120〉endogenous that is used for the human body pathogenic agent with reference to and use
<130>042146
<160>35
<170>PatentIn version 3.1
<210>1
<211>1812
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
ataaccatgc acactactat aaccacccta accctgactt ccctaattcc ccccatcctt 60
accaccctcg ttaaccctaa caaaaaaaac tcataccccc attatgtaaa atccattgtc 120
gcatccacct ttattatcag tctcttcccc acaacaatat tcatgtgcct agaccaagaa 180
gttattatct cgaactgaca ctgagccaca acccaaacaa cccagctctc cctaagcttc 240
aaactagact acttctccat aatattcatc cctgtagcat tgttcgttac atggtccatc 300
atagaattct cactgtgata tataaactca gacccaaaca ttaatcagtt cttcaaatat 360
ctactcattt tcctaattac catactaatc ttagttaccg ctaacaacct attccaactg 420
ttcatcggct gagagggcgt aggaattata tccttcttgc tcatcagttg atgatacgcc 480
cgagcagatg ccaacacagc agccattcaa gcagtcctat acaaccgtat cggcgatatc 540
ggtttcatcc tcgccttagc atgatttatc ctacactcca actcatgaga cccacaacaa 600
atagcccttc taaacgctaa tccaagcctc accccactac taggcctcct cctagcagca 660
gcaggcaaat cagcccaatt aggtctccac ccctgactcc cctcagccat agaaggcccc 720
accccagtct cagccctact ccactcaagc actatagttg tagcaggaat cttcttactc 780
atccgcttcc accccctagc agaaaatagc ccactaatcc aaactctaac actatgctta 840
ggcgctatca ccactctgtt cgcagcagtc tgcgccctta cacaaaatga catcaaaaaa 900
atcgtagcct tctccacttc aagtcaacta ggactcataa tagttacaat cggcatcaac 960
caaccacacc tagcattcct gcacatctgt acccacgcct tcttcaaagc catactattt 1020
atgtgctccg ggtccatcat ccacaacctt aacaatgaac aagatattcg aaaaatagga 1080
ggactactca aaaccatacc tctcacttca acctccctca ccattggcag cctagcatta 1140
gcaggaatac ctttcctcac aggtttctac tccaaagacc acatcatcga aaccgcaaac 1200
atatcataca caaacgcctg agccctatct attactctca tcgctacctc cctgacaagc 1260
gcctatagca ctcgaataat tcttctcacc ctaacaggtc aacctcgctt ccccaccctt 1320
actaacatta acgaaaataa ccccacccta ctaaacccca ttaaacgcct ggcagccgga 1380
agcctattcg caggatttct cattactaac aacatttccc ccgcatcccc cttccaaaca 1440
acaatccccc tctacctaaa actcacagcc ctcgctgtca ctttcctagg acttctaaca 1500
gccctagacc tcaactacct aaccaacaaa cttaaaataa aatccccact atgcacattt 1560
tatttctcca acatactcgg attctaccct agcatcacac accgcacaat cccctatcta 1620
ggccttctta cgagccaaaa cctgccccta ctcctcctag acctaacctg actagaaaag 1680
ctattaccta aaacaatttc acagcaccaa atctccacct ccatcatcac ctcaacccaa 1740
aaaggcataa ttaaacttta cttcctctct ttcttcttcc cactcatcct aaccctactc 1800
ctaatcacat aa 1812
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>2
ggctgagagg gcgtaggaat t 21
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>3
tacgcccgag cagatgccaa ca 22
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>4
gcgaggatga aaccgatatc g 21
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>5
tcggctgaga gggcgtagga at 22
<210>6
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>6
tacgcccgag cagatgccaa ca 22
<210>7
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>7
ggcgaggatg aaaccgatat cg 22
<210>8
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>8
atcggctgag agggcgtagg aa 22
<210>9
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>9
tgatacgccc gagcagatgc ca 22
<210>10
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>10
ggcgaggatg aaaccgatat cg 22
<210>11
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>11
tccaactgtt catcggctga ga 22
<210>12
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>12
tacgcccgag cagatgccaa ca 22
<210>13
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>13
catgctaagg cgaggatgaa ac 22
<210>14
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>14
tgccaagtgt ttgctga 17
<210>15
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>15
aagaagggga cggtaga 17
<210>16
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>16
acgcaacccc cactggctgg gg 22
<210>17
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>17
taggacccct gctcgtg 17
<210>18
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>18
atgggatggg aatacaag 18
<210>19
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>19
aggcggggtt tttcttgttg a 21
<210>20
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>20
gcacttgtat tcccatcc 18
<210>21
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>21
aaagccctac gaaccac 17
<210>22
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>22
atcccatcat cctgggcttt cg 22
<210>23
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>23
tgccaagtgt ttgctga 17
<210>24
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>24
agaaggggac ggtagatc 18
<210>25
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>25
ccactggctg gggcttggcc att 23
<210>26
<211>341
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉HBV positive control sequence
<400>26
gccagccccc gatcgggggc gacactccac catagatcac tcccctgtga ggaactactg 60
tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggcc 120
cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag 180
gacgaccggg tcctttcttg gatctacccg ctcaatgcct ggagatttgg gcgtgccccc 240
gcgagaccgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300
gtgcttgcga gtgccccggg aggtctcgta gaccgtgcat c 341
<210>27
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>27
tggtttgtcc gcatcatggt 20
<210>28
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>28
agtccatgat gaagaaccaa 20
<210>29
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>29
tgacgggcta tatgtggatc 20
<210>30
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>30
ggttggaagc cgtgaagctg 20
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>31
ggttcagtga cagtgagcta 20
<210>32
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>32
ttgagcgtgt gagatagtgc ag 22
<210>33
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>33
tgatggatcg atgacagtcg 20
<210>34
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>34
gtgtaagtca gtgatagcgt 20
<210>35
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>35
gtgtatgact gatcgcgtga gt 22
Claims (10)
1. a human body endogenous dna contrast is characterized in that, it has the nucleotide sequence shown in the SEQ ID NO:1.
2. the purposes of human body endogenous dna contrast as claimed in claim 1 is characterized in that, it is used as the object of reference of the DNA when increasing human body pathogen in same amplification system from biological sample.
3. purposes as claimed in claim 2 is characterized in that, in described amplification system, contain be selected from the amplification this human body endogenous dna contrast primer to with amplified production bonded probe.
4. purposes as claimed in claim 3 is characterized in that, described primer to probe be selected from following each the group:
Group (a):
Upstream primer 1 GGCTGAGAGGGCGTAGGAATT (SEQ ID NO:2)
Downstream primer 1 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:3)
Probe 1 GCGAGGATGAAACCGATATCG (SEQ ID NO:4)
Group (b):
Upstream primer 2TCGGCTGAGAGGGCGTAGGAAT (SEQ ID NO:5)
Downstream primer 2 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:6)
Probe 2 GGCGAGGATGAAACCGATATCG (SEQ ID NO:7)
Group (c):
Upstream primer 3 ATCGGCTGAGAGGGCGTAGGAA (SEQ ID NO:8)
Downstream primer 3 TGATACGCCCGAGCAGATGCCA (SEQ ID NO:9)
Probe 3 GGCGAGGATGAAACCGATATCG (SEQ ID NO:1O)
Group (d):
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 CATGCTAAGGCGAGGATGAAAC (SEQ ID NO:13).
5. purposes as claimed in claim 2 is characterized in that described human body pathogen comprises bacterium, chlamydozoan, mycoplasma or dna virus.
6. purposes as claimed in claim 2 is characterized in that wherein said biological sample is selected from human blood, serum, blood plasma, saliva, cerebrospinal fluid, somatocyte.
7. a method that is used for vitro detection pathogenic agent DNA is characterized in that, described method comprises the following steps:
(a) with the human body pathogen DNA that derives from described sample and human body endogenous crt gene DNA as template, with first primer of specific amplification human body pathogen DNA to second primer of specific amplification human body endogenous crt gene DNA to as Oligonucleolide primers, carry out nucleic acid amplification reaction, thereby produce first specific amplification products of human body pathogen DNA and second specific amplification products of human body endogenous crt gene DNA;
(b) whether the existence of described first specific amplification products of detection and second specific amplification products;
Wherein, the existence of described two specific specificity amplified productions just shows that human body pathogen DNA and human body endogenous crt gene DNA are present in the described sample; Second specific amplification products that does not have human body endogenous crt gene DNA just represents that detected result is a false negative.
8. detection kit, it is characterized in that, it is right that it contains the primer of the described human body endogenous dna of specific amplification claim 1 contrast, described primer length is a 15-35 Nucleotide, and the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1, and the length of amplified production is 100-1000bp.
9. test kit as claimed in claim 8 is characterized in that, described primer to probe be selected from following each the group:
Group (a):
Upstream primer 1 GGCTGAGAGGGCGTAGGAATT (SEQ ID NO:2)
Downstream primer 1 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:3)
Probe 1 GCGAGGATGAAACCGATATCG (SEQ ID NO:4)
Group (b):
Upstream primer 2 TCGGCTGAGAGGGCGTAGGAAT (SEQ ID NO:5)
Downstream primer 2 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:6)
Probe 2 GGCGAGGATGAAACCGATATCG (SEQ ID NO:7)
Group (c):
Upstream primer 3 ATCGGCTGAGAGGGCGTAGGAA (SEQ ID NO:8)
Downstream primer 3 TGATACGCCCGAGCAGATGCCA (SEQ ID NO:9)
Probe 3 GGCGAGGATGAAACCGATATCG (SEQ ID NO:10)
Group (d):
Upstream primer 4 TCCAACTGTTCATCGGCTGAGA (SEQ ID NO:11)
Downstream primer 4 TACGCCCGAGCAGATGCCAACA (SEQ ID NO:12)
Probe 4 CATGCTAAGGCGAGGATGAAAC (SEQ ID NO:13).
10. test kit as claimed in claim 9 is characterized in that, also contain the Oligonucleolide primers that detects hepatitis B virus DNA to and probe, described primer is to being selected from down group with probe:
Group (e):
Upstream primer 5 TGCCAAGTGTTTGCTGA (SEQ ID NO:14)
Downstream primer 5 AAGAAGGGGACGGTAGA (SEQ ID NO:15)
Probe 5 ACGCAACCCCCACTGGCTGGGG (SEQ ID NO:16)
Group (f):
Upstream primer 6 TAGGACCCCTGCTCGTG (SEQ ID NO:17)
Downstream primer 6 ATGGGATGGGAATACAAG (SEQ ID NO:18)
Probe 6 AGGCGGGGTTTTTCTTGTTGA (SEQ ID NO:19)
Group (g):
Upstream primer 7 GCACTTGTATTCCCATCC (SEQ ID NO:20)
Downstream primer 7 AAAGCCCTACGAACCAC (SEQ ID NO:21)
Probe 7 ATCCCATCATCCTGGGCTTTCG (SEQ ID NO:22)
Group (h):
Upstream primer 8 TGCCAAGTGTTTGCTGA (SEQ ID NO:23)
Downstream primer 8 AGAAGGGGACGGTAGATC (SEQ ID NO:24)
Probe 8 CCACTGGCTGGGGCTTGGCCATT (SEQ ID NO:25);
Perhaps also contain the Oligonucleolide primers that detects ureaplasma urealyticum DNA to and probe, described primer is to being selected from down group with probe:
Upstream primer: 5 '-TGGTT TGTCC GCATC ATGGT-3 ' (SEQ ID NO:27)
Downstream primer; 5 '-AGTCC ATGAT GAAGA ACCAA-3 ' (SEQ ID NO:28)
Probe: FAM-TGACG GGCTA TATGT GGATC-TAMRA (SEQ ID NO:29);
Perhaps also contain the Oligonucleolide primers that detects gonococcus DNA to and probe, described primer is to being selected from down group with probe:
Upstream primer: 5 '-GGTTG GAAGC CGTGA AGCTG-3 ' (SEQ ID NO:30)
Downstream primer; 5 '-GGTTC AGTGA CAGTG AGCTA-3 ' (SEQ ID NO:31)
Probe: FAM-TTGAG CGTGT GAGAT AGTGC AG-TAMRA (SEQ ID NO:32);
Perhaps also contain the Oligonucleolide primers that detects the former DNA of trachoma clothing to and probe, described primer is to being selected from down group with probe:
Upstream primer: 5 '-TGATG GATCG ATGAC AGTCG-3 ' (SEQ ID NO:33)
Downstream primer; 5 '-GTGTA AGTCA GTGAT AGCGT-3 ' (SEQ ID NO:34)
Probe: FAM-GTGTA TGACT GATCG CGTGA GT-TAMRA (SEQ ID NO:35).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100893718A CN100562583C (en) | 2004-12-10 | 2004-12-10 | Be used for the endogenous reference and the application thereof of human body pathogenic agent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100893718A CN100562583C (en) | 2004-12-10 | 2004-12-10 | Be used for the endogenous reference and the application thereof of human body pathogenic agent |
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| Publication Number | Publication Date |
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| CN1786193A true CN1786193A (en) | 2006-06-14 |
| CN100562583C CN100562583C (en) | 2009-11-25 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851661A (en) * | 2010-05-10 | 2010-10-06 | 其昌達生物高科技(上海)有限公司 | Method for shortening mycoplasma clinical detection time under A-PEG-B array |
| CN102559861A (en) * | 2010-12-30 | 2012-07-11 | 上海复星医学科技发展有限公司 | Kit for rapidly detecting nucleic acid of chlamydia trachomatis (CT) |
-
2004
- 2004-12-10 CN CNB2004100893718A patent/CN100562583C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851661A (en) * | 2010-05-10 | 2010-10-06 | 其昌達生物高科技(上海)有限公司 | Method for shortening mycoplasma clinical detection time under A-PEG-B array |
| CN101851661B (en) * | 2010-05-10 | 2015-09-09 | 其昌達生物高科技(上海)有限公司 | The method of the detection mycoplasma time of accelerating under A-PEG-B array of non-diagnostic object |
| CN102559861A (en) * | 2010-12-30 | 2012-07-11 | 上海复星医学科技发展有限公司 | Kit for rapidly detecting nucleic acid of chlamydia trachomatis (CT) |
| CN102559861B (en) * | 2010-12-30 | 2016-08-10 | 上海星耀医学科技发展有限公司 | Chlamydia trachomatis nucleic acid quick detection kit |
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| Publication number | Publication date |
|---|---|
| CN100562583C (en) | 2009-11-25 |
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