CN103866013A - Orientia tsutsugamushi disease nucleic acid detection kit - Google Patents
Orientia tsutsugamushi disease nucleic acid detection kit Download PDFInfo
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- CN103866013A CN103866013A CN201410079165.2A CN201410079165A CN103866013A CN 103866013 A CN103866013 A CN 103866013A CN 201410079165 A CN201410079165 A CN 201410079165A CN 103866013 A CN103866013 A CN 103866013A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to an orientia tsutsugamushi disease nucleic acid detection kit, and in particular relates to a kit for detecting orientia tsutsugamushi disease nucleic acid by using a composite probe real-time fluorescence PCR (Polymerase Chain Reaction) technique. The kit is used for quantitatively detecting an orientia tsutsugamushi disease in a sample, and can be applied to assistant diagnosis on orientia tsutsugamushi disease infection.
Description
Technical field
The present invention relates to Orientia Tsutsugamushi kit for detecting nucleic acid, particularly relate to a kind of test kit that utilizes combined probe technology real-time fluorescence PCR technology for detection Orientia Tsutsugamushi.This test kit carries out qualitative detection by the Orientia Tsutsugamushi in sample, can be applicable to the auxiliary diagnosis infecting containing Orientia Tsutsugamushi.
Background technology
Orientia Tsutsugamushi (Orientia tsutsugamushi) is to be bitten and propagated and cause and the pathogenic agent of tsutsugamushi fever claim in the past Rickettsia tsutsugamushi by Mite larva.Take muroid as main contagium, bite and propagate by Mite larva.Orientia Tsutsugamushi is rounded, ellipse or rod-short.Gram's staining feminine gender.Giemsa staining is red-purple.At fb dur, can isolate pathogenic agent from patient's eschar, blood, lymphoglandula or marrow etc.Rickettsia tsutsugamushi has nature bursting tendency, is difficult for preserving.All very sensitive to heat and general sterilization method, stronger to the resistibility of low temperature.Rickettsia tsutsugamushi in different areas, the antigenicity between homophyletic does not have larger difference, also not identical to people's virulence.Take heating, eschar or ulcer, lymphadenectasis and fash as feature, easily cause mistaken diagnosis clinically.[Ruang-areerate?T，Jeamwattanalert?P，Rodkvamtook?W，et?al.Genotype?divetsity?and?distribution?of?Orientia?tsutsugamushi?causing?scrub?typhus?in?Thailand[J].Journal?of?clinical?microbiology，2011，49(7)：2584-2589；Luksameetanasan?R，Blacksell?S?D，Kalambaheti?T，et?al.Patient?and?sample-related?factots?that?effect?the?success?of?in?vitro?isolation?of?Orientia?tsutsugamushi[J].2007.]。
In order effectively to diagnose and prevent tsutsugamushi fever, both at home and abroad the major antigen molecule of Orientia Tsutsugamushi is conducted in-depth research.Studying more is 56kD, 47kD, 58kD, 110kD and 22kD etc.58kD albumen is thalline outer membrane protein, high conservative, and each strain is total, is Orientia Tsutsugamushi genus-specific antigen, is also potentiality protective antigen.56kD albumen is thalline major outer membrane albumen, is exposed to thalline surface, with rickettsial absorption with invade relevant.This antigen contains type specific antigen and determines family, also can produce Idiotype provide protection.22kD albumen is also containing type specific antigen epi-position.47kDa albumen is one of albumen of Orientia tsutsugamushi surface rich content, has stronger immunogenicity.Because this albumen is for belonging to specificity, with respect to the 56kDa albumen of type specificity, it has potential superiority for diagnosing and preventing aspect infection of different type Orientia Tsutsugamushi.[Seong?S?Y，Huh?M?S，Jang?W?J，et?al.Induction?of?homologous?immune?response?to?Rickettsia?tsutsugamushi?Boryong?with?a?partial56-ki?lodalton?recombinant?antigen?fused?with?the?maltose-binding?protein?MBP-Bor56[J].Infection?and?immunity，1997，65(4)：1541-1545.；Jiang?J，Chan?T?C，Temenak?J?J，et?al.Development?of?a?quantitative?real-time?polymerase?chain?reaction?assay?specific?for?Orientia?tsutsugamushi[R].NAVAL?MEDICAL?RESEARCH?CENTER?SILVER?SPRING?MD，2004.]。
Clinical microorganism laboratory is to comparatively difficulty of the detection of Orientia Tsutsugamushi, and major cause comprises: 1) serodiagnosis is difficult to detect antibody in morbidity in one week, and pathogen separation complicated operation, and separation rate is very low; 2) etiological examination length consuming time exists and causes at any time the danger that people is contaminted in separation operation process in addition; 3) may occurring of the many types of and multiple complications of the hot clinical manifestation of Q, bring certain difficulty to clinical diagnosis.Now the main method that detects clinically at present Orientia Tsutsugamushi is described below:
1. blood picture white blood cell count(WBC) reduces or is normal.While having other complication, can increase, classification is often neutrophilic leukocytosis, shift to left (shaft-like neutrophilic leukocytosis).
2. pyrogenic stage blood samples of patients 0.5～1mL is got in etiological examination, is inoculated in mouse peritoneal.If mouse fell ill in 1～2 week, death, get its peritonaeum, mesentery or spleen section and on slide glass, make printingout, after doing, dye through Ji's nurse Sa, then use microscope oil spectroscopy.The Orientia Tsutsugamushi that visible red-purple, cluster distribute in the kytoplasm of monocyte, scavenger cell.
3. Serological testing has larger value to the diagnosis of this disease.Commonly use and have following four kinds of methods.
(1) outer striking reaction (Bacillus proteus OX19 agglutination test): to Bacillus proteus OXK antigen generation agglutination reaction, and tire 1: 160 or more than, or early, late period paired sera tire and be 4 times and increase above elder and have diagnostic significance.
(2) complement fixation test (CFT): apply local representative strains antigen or polyvalent antigen and detect, specific degree is strong, and recall rate is higher.
(3) indirect immunofluorescence assay: detect specific IgM, IgG antibody in patients serum.There is higher susceptibility, specificity and circulation ratio, be regarded as " gold standard ".
3. molecular diagnosis method: easy fast, and extremely sensitive, solved that tsutsugamushi fever patient their early stage antibody titers is too low cannot carry out a SD difficult problem, be the valuable method of tsutsugamushi fever early diagnosis.Wherein, real-time fluorescence PCR rule be generally acknowledged bacterial detection the most accurately, method the most efficiently.Also the detection that has at present several PCR-based ends and real-time detection technique to be used to produce KPC bacterial strain, these technology have higher specificity, but need technical professional and equipment to implement, and testing cost is relative also higher.
This test is take Orientia Tsutsugamushi 47KD gene as target sequence, and application complex probe has been set up a kind of method of special, sensitive, rapid detection Orientia Tsutsugamushi.
Summary of the invention
The object of the present invention is to provide a kind of real-time fluorescence PCR method to detect the test kit (being called for short test kit below) of Orientia Tsutsugamushi nucleic acid, this test kit comprises: (1) sample treatment solution A, sample treatment solution B, PCR reaction solution A, PCR reaction solution B, negative control, strong sun contrast, weak positive control.(2) separate and concentrate the packing box of packing these reagent bottles or pipe.
In order to solve above-mentioned task, concrete technological line of the present invention is:
(1) Oligonucleolide primers and the oligonucleotide probe that can be combined with target polynucleotide that design for Orientia Tsutsugamushi gene conserved sequence.
(2) oligonucleotide probe mark fluorescent generation group, makes said fluorescence generation group and the indirect combination of target polynucleotide being amplified.
(3) be suitable for reaction system and the fluorescent detection system of pcr amplification.Nucleic acid amplification reaction system comprise hot resistant DNA polymerase, 2 '-deoxynucleoside triphosphate, can with the forward primer of the Article 1 chain combination of double-stranded target polynucleotide, can be with the reverse primer of the Article 2 chain combination of double-stranded target polynucleotide, can be combined with target polynucleotide and two ends are combined with respectively the oligonucleotide probe of fluorescence generation group and fluorescent quenching group, the damping fluid that contains magnesium ion and methane amide.
(4) from sample to be tested, extract DNA, add in aforesaid reaction system, directly through pcr amplification, judge whether to exist Orientia Tsutsugamushi from amplified fluorescence curve.
A preferred embodiment according to the present invention, wherein the PCR reaction solution of test kit is made up of 10 × PCR damping fluid, target gene forward primer, reverse primer, oligonucleotide probe, aqua sterilisa, it is characterized in that for forward and the reverse primer of target polynucleotide amplification be respectively 5 '-AACTGATTTTATTCAAACTAATGCT-3 ' and 5 '-AGCAAAACTTATGCCTGAGTAA-3 '; Oligonucleotide probe for target polynucleotide amplification and monitoring system is f:FAM-TGGTCCAGTTAACCAAAATGCTCTT-P, q:GATTAAACATCGGTCCACCAAA-dabcyl; PCR reaction enzymes system comprises Taq enzyme and UNG enzyme.
Another preferred embodiment according to the present invention, negative quality control product is aqua sterilisa; Positive quality control product is, by vitro recombination, object fragment gene is imported to T carrier, builds and extract recombinant plasmid dna, and with TE liquid, dilution-20C preserves.For the actual quality control detecting.
According to test kit provided by the invention, Orientia Tsutsugamushi is detected, the method comprises the following steps:
(1) carry out the DNA extraction of positive and negative quality control product and sample to be tested with sample treatment solution.Sample treatment solution, except the method that the present invention mentions, can also use other ripe DNA extraction method and test kit.
(2) DNA of extraction is joined in the PCR reaction tubes that contains PCR reaction solution and PCR reaction enzymes system, carry out pcr amplification, use fluorescent quantitative PCR detector to detect.
Singularity during test kit of the present invention detects for Orientia Tsutsugamushi, consumption, annealing temperature etc. to primer concentration and probe concentration, Taq enzyme in reaction system are all optimized, combined with fluorescent round pcr, for Orientia Tsutsugamushi detection of nucleic acids, and develop for Orientia Tsutsugamushi kit for detecting nucleic acid, sensitivity can reach 10
2copy/ml.
The present invention compared with prior art, has following advantages:
(1) the combined probe technology adopting is domestic unique real-time fluorescence PCR technology (patent No.: ZL99125469.4, relevant paper is published in Anal Biochem, 2002,309:206-211), is to have independent intellectual property right product.Specificity is stronger, and sensitivity is higher;
(2) totally-enclosed reaction, extracts after DNA of bacteria, is directly used in PCR and detects, and has avoided polluting occurring;
(3) detection speed is fast, and whole process is no more than 2 hours;
(4) simple to operate, controllability is strong, can carry out batch samples detection, is conducive to industrialization.
Accompanying drawing explanation
Fig. 1 visualizingre agent box detects the condition setting of Orientia Tsutsugamushi nucleic acid.
The amplification curve of different concns sample when Fig. 2 visualizingre agent box detects Orientia Tsutsugamushi nucleic acid, the amplification curve of two positive samples is S shape, all can be judged to be the positive.
The amplification curve of ten ' negative ' specimens of Fig. 3 visualizingre agent box, does not all have a S shape feature, can be judged to be feminine gender.
Fig. 4 shows the amplification curve of strong positive contrast and weak positive control.Diagram amplification curve is S type curve, and the detection system Orientia Tsutsugamushi nucleic acid that effectively increased is described.
Fig. 5 shows negative quality control product amplification curve.Diagram amplification curve is put down as straight broken line, there is no intersection point, and curve is not S-type, and the pollution that there is no Orientia Tsutsugamushi nucleic acid in testing process is described with fluoroscopic examination threshold line.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Orientia Tsutsugamushi kit for detecting nucleic acid detection method
1. the collection of sample
A) gather serum: get person under inspection's venous blood 2ml, be collected in aseptic centrifuge tube, room temperature is placed and is no more than 4 hours, centrifugal 20 minutes of 3000rpm, draws serum and proceeds in another aseptic centrifuge tube for subsequent use;
B) gather blood plasma: go person under inspection's venous blood 2ml in the aseptic centrifuge tube that contains antithrombotics (banning use of anticoagulant heparin), room temperature is placed and is no more than 4 hours, centrifugal 20 minutes of 3000rpm, separated plasma, proceeds to aseptic centrifuge tube for subsequent use.
2. nucleic acid extraction
2.1 nucleic acid extraction
A) draw 100 μ l sample preparation liquid A to centrifuge tube with band filter core suction nozzle.
B) add 100 μ l samples to be tested.
C) lid upper tube cap, concussion mixes, centrifugal 10 minutes of 1,3000rpm.
D) inhale and abandon 175 μ l supernatants (avoiding stirring or encountering precipitation).
E) add 25 μ l sample preparation liquid B.
F) fully concussion mixes, and after the centrifugal several seconds of low speed (2000rpm), 100 ℃ dry bathes or boiling water bath 10 minutes.
G) 1, centrifugal 10 minutes of 3000rpm, getting supernatant is PCR reaction template (supernatant, if do not used immediately, need be put-20 ℃ of preservations, needs centrifugal 10 minutes of 1,3000rpm while reusing).
3.PCR detects
3.1 preparation systems
A) get a 0.5ml centrifuge tube, prepare PCR reaction solution (N is reaction tubes number) by following composition:
PCR reaction solution A26 μ l × (N+1)
PCR reaction solution B1 μ l × (N+i)
Be filled in PCR reaction tubes by 27.0 μ l/ pipes point.
(noting: before use, guarantee that PCR reaction solution fully dissolves, PCR reaction solution B needs centrifugal to guarantee that all enzymes concentrate on bottom before use)
B) application of sample
The sample supernatant liquor that 3 μ l were processed adds respectively and in PCR reaction tubes, covers tightly pipe lid, after the centrifugal 1min of 6000rpm, puts into pcr amplification instrument.
Amplification program arranges
The parameter of Bio-Rad, ABI7300/7500PCR amplification instrument arranges as follows
Instrument sense channel is selected: FAM is RM amplified signal.
The parameter of Roche LightCycler2.0PCR amplification instrument arranges as follows
Fluorescence channel is selected: 530 is RM amplified signal.
4. interpretation of result
After reaction finishes, analyze according to the software of each quasi-instrument, regulate noise margin to more than baseline noise, threshold setting principle is as the criterion just above the vertex of normal negative control curve (random noise line) with threshold line.Baseline is chosen 6～10 or 6～15 race ways.Make negative control Ct value not occur any numerical value as far as possible.
5. result is judged
If sample increases without S type curve, interior mark result is positive simultaneously, judges that this sample is negative;
If sample amplification curve is obvious S type, be judged to be positive.On this basis, if Ct value is greater than 20, judge that sample, as the general positive, is labeled as "+"; If Ct value is less than or equal to 20, be judged to be strong positive, be labeled as " ++ ".
The use of quality control product in Orientia Tsutsugamushi kit for detecting nucleic acid
In Orientia Tsutsugamushi kit for detecting nucleic acid, quality control product comprises strong positive contrast, weak positive control, negative quality control product, and for clinical trial quality control, working method is with sample to be checked.
Quality control standard
Negative control result is negative, and Ct value is 0 or blank, and interior mark result is positive simultaneously;
Strong positive results of comparison is positive, Ct value≤23;
Weak positive control result is positive, Ct value≤33;
Negative control, positive control result need meet in same once experiment simultaneously, otherwise this experiment is invalid, and all experiments were should re-start.
Claims (4)
1. the test kit (being called for short test kit below) of a real-time fluorescence PCR method detection Orientia Tsutsugamushi nucleic acid, this test kit comprises: (1) sample treatment solution A, sample treatment solution B, PCR reaction solution A, PCR reaction solution B, negative control, strong positive contrast, weak positive control; (2) separate and concentrate the packing box of packing these reagent bottles or pipe.Wherein the PCR reaction solution of test kit is made up of forward primer, reverse primer, oligonucleotide probe and the aqua sterilisa of 10 × PCR damping fluid, target gene, it is characterized in that for forward and the reverse primer of target polynucleotide amplification be respectively 5 '-AACTGATTTTATTCAAACTAATGCT-3 ' and 5 '-AGCAAAACTTATGCCTGAGTAA-3 '.
2. according to the test kit of claim 1, be further characterized in that for the oligonucleotide probe of target polynucleotide amplification be 5 '-FAM-TGGTCCAGTTAACCAAAATGCTCTT-P-3 ' and 5 '-GATTAAACATCGGTCCACCAAA-dabcy1-3 '.
3. according to the test kit of claim 1, be further characterized in that PCR reaction enzymes system comprises Taq enzyme and UNG enzyme.
4. according to the test kit of claim 1, be further characterized in that negative quality control product is aqua sterilisa; Positive quality control product is, by vitro recombination, object fragment gene is imported to pGEM-T carrier, builds and extract recombinant plasmid dna.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104975095A (en) * | 2015-07-14 | 2015-10-14 | 宋锋林 | Real-time fluorescence quantification PCR detection system and method by using TaqMan-MGB probe for detecting orientia tsutsugamushi |
| CN105548178A (en) * | 2014-10-23 | 2016-05-04 | 免疫制药公司 | Fluorescence reader of medical diagnostic kits |
| CN108070638A (en) * | 2017-09-20 | 2018-05-25 | 李佳萌 | A kind of recombinase polymerase constant-temperature amplification method, its primer special and probe and purposes for detecting Orientia Tsutsugamushi |
| CN108474797A (en) * | 2015-12-30 | 2018-08-31 | 仁荷大学校产学协力团 | Interferon by detecting peripheral blood mononuclear cells is secreted come the diagnostic method of diagnosis of tsutsugamushi disease |
| CN111712583A (en) * | 2018-01-08 | 2020-09-25 | 朝鲜大学校产学协力团 | Method for diagnosing tsutsutsugamushi disease using multi-copy gene |
| CN115232798A (en) * | 2022-08-08 | 2022-10-25 | 南京工业大学 | Hybridoma cell strain 5B3, tsutsugamushi oriental 56kDa protein monoclonal antibody, preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548178A (en) * | 2014-10-23 | 2016-05-04 | 免疫制药公司 | Fluorescence reader of medical diagnostic kits |
| CN104975095A (en) * | 2015-07-14 | 2015-10-14 | 宋锋林 | Real-time fluorescence quantification PCR detection system and method by using TaqMan-MGB probe for detecting orientia tsutsugamushi |
| CN108474797A (en) * | 2015-12-30 | 2018-08-31 | 仁荷大学校产学协力团 | Interferon by detecting peripheral blood mononuclear cells is secreted come the diagnostic method of diagnosis of tsutsugamushi disease |
| CN108070638A (en) * | 2017-09-20 | 2018-05-25 | 李佳萌 | A kind of recombinase polymerase constant-temperature amplification method, its primer special and probe and purposes for detecting Orientia Tsutsugamushi |
| CN108070638B (en) * | 2017-09-20 | 2021-06-08 | 李佳萌 | Recombinase polymerase isothermal amplification method for detecting orientia tsutsutsugamushi, special primer and probe thereof and application |
| CN111712583A (en) * | 2018-01-08 | 2020-09-25 | 朝鲜大学校产学协力团 | Method for diagnosing tsutsutsugamushi disease using multi-copy gene |
| CN111712583B (en) * | 2018-01-08 | 2023-10-31 | 朝鲜大学校产学协力团 | Method for diagnosing tsutsugamushi disease using multiple copy genes |
| CN115232798A (en) * | 2022-08-08 | 2022-10-25 | 南京工业大学 | Hybridoma cell strain 5B3, tsutsugamushi oriental 56kDa protein monoclonal antibody, preparation method and application thereof |
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Application publication date: 20140618 |