CN108642196A - Ring mediated isothermal amplification detects the kit and method of mycoplasma pneumoniae - Google Patents
Ring mediated isothermal amplification detects the kit and method of mycoplasma pneumoniae Download PDFInfo
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- CN108642196A CN108642196A CN201810704561.8A CN201810704561A CN108642196A CN 108642196 A CN108642196 A CN 108642196A CN 201810704561 A CN201810704561 A CN 201810704561A CN 108642196 A CN108642196 A CN 108642196A
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses the kits that a kind of ring mediated isothermal amplification detects mycoplasma pneumoniae, and the kit includes primer sets, and the primer sets are by SEQ ID NO:Upstream outer primers F 3 shown in 1, SEQ ID NO:Downstream outer primer B3, SEQ ID NO shown in 2:Upstream internal primers F IP shown in 3 and SEQ ID NO:Downstream inner primer BIP shown in 4 is formed.The kit of the present invention has the characteristics that high specificity, high sensitivity, and detection mycoplasma pneumoniae is convenient and efficient, cheap, is promoted the use of suitable for different medical unit.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of ring mediated isothermal amplification detection mycoplasma pneumoniae
Kit and method.
Background technology
Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) is a kind of acellular between virus and bacterium
Wall-shaped microorganism, and cause the important pathogen body of community-acquired pneumonia in children, there are case appearance in the four seasons, but with summer and autumn
Based on morbidity.Disease in school age children is more common in MP infection, accounts for about 19.1-26% in because of respiratory tract infection Hospitalized Children.In addition to drawing
It rises outside pulmonary infection, MP can also cause a variety of extrapulmonary complications, so as to cause the damage of multiple organ dysfunction.
Pneumonia early clinical manifestation, imageological examination etc. caused by MP usually lack specificity, it is difficult to pass through clinical symptoms
It is diagnosed with sign, and MP needs to be controlled using macrolide antibiotics to beta-lactam enzyme antibiotic ineffective
It treats, MP infection is also possible to cause some serious clinical syndromes, therefore accurate and the infection of quick diagnosis MP is most important.
The test in laboratory method of MP includes MP cultures, Serologic detection and nucleic acid amplification technologies (NAAT).Cultivation is
The safe criterion of MP infection is diagnosed, the clinical separation strain of MP is also most important to monitoring its epidemiological trends, but MP growths are slow
Slowly, it usually needs can just see within 2 weeks or more as a result, and culture positive rate it is low, therefore be not suitable for routine clinical detection.Serology
Detection is the most extensive MP detection methods of current clinical application, including gelatin particle agglutination experiment, enzyme-linked immunosorbent assay, serum
The experiment of condensation collection and complement fixation test etc..
Human infection MP will produce specific IgM, IgA and IgG class antibody, and convalescence and acute stage MP antibody titers are in 4
When again or 4 times or more are increased or lowered, it can be diagnosed as MP infection, but clinically more difficult acquisition paired sera is detected, single part
Serum result tends not to truly reflect infant infection conditions.Although, earlier than IgG antibody, exempting from occurs in serum IgM class antibody
The low infant of epidemic disease power or 2 years old infant below be not because developing immune system is complete, and antibody does not generate so as to cause vacation after infecting MP
It is negative.
With the fast development of molecular biology, the important method that NAAT is gradually infected through becoming Rapid＆Early diagnosis MP,
It has many advantages, such as high sensitivity, high specificity, and not infected time restriction, is not also influenced by infant immune function, right
It is significant in the early diagnosis of MP infection.NAAT includes mainly that PCR methods, real-time fluorescence PCR method, multiplex PCR and ring mediate
Isothermal duplication (LAMP) etc., PCR methods need expensive PCR cycle instrument, and more demanding to operating personnel's technical merit, it is difficult to
In different medical unit extensive use.Report within 2000 a kind of new nucleic acid amplification method-ring mediated isothermal amplification
(LAMP), principle is to use the archaeal dna polymerase with strand-displacement activity and one group of primer specially designed under constant temperature
Quickly and efficiently expand target DNA.Compared with traditional detection method, 4 primers of LAMP are according to 6 of target sequence not same districts
Domain design, there is high degree of specificity, once there is primer mismatch that reaction will be caused not occur.In addition, PCR is compared in LAMP detections
10-100 times of high sensitivity can detect 10 copies even less template.Importantly, LAMP reaction costs are cheap, behaviour
Make simple, it is only necessary to which the simple devices such as water-bath can complete reaction.LAMP amplifications can directly be looked by using dyestuff
It sees, so as to shorten detection time.
The existing kit that MP is detected using LAMP method currently on the market, but it needs to react 60min, and need examining
Reaction tube addition fluorescent dye SYBR Green observations color change is opened after the completion of surveying to uncap since LAMP sensitivitys are high
After be difficult to avoid that Aerosol Pollution occur, it is also necessary in ultraviolet lamp apparatus, it is therefore desirable to a kind of faster whole stopped pipe
The method that LAMP detects MP, direct visual color variation, to reduce the detection need for polluting and meeting different medical unit
It asks.
Invention content
The present invention for mycoplasma pneumoniae detection speed in the prior art, ask by slow, of high cost and technology complicated for operation
Topic is, and it is an object of the present invention to provide a kind of ring mediated isothermal amplification detects the kit of mycoplasma pneumoniae, to meet different medical unit
Detection demand.
The present invention ring mediated isothermal amplification detection mycoplasma pneumoniae kit include primer sets, the primer sets by
SEQ ID NO:Upstream outer primers F 3 shown in 1, SEQ ID NO:Downstream outer primer B3, SEQ ID NO shown in 2:3 institutes
The upstream internal primers F IP and SEQ ID NO shown:Downstream inner primer BIP shown in 4 is formed.
Specifically, upstream outer primers F 3:5'-CTGGCCAATCCACCCAAC-3'；
Downstream outer primer B3:5'-ACTGGAAAGGGCAGTACCA-3'；
Upstream internal primers F IP:5'-ACCATTGTCCTCCGAGTCCGATGTCAGGGGACACCAAAGTC-3'；
Downstream inner primer BIP:5'-GATCTCGCCAACGCTCCCATAAATCGTCCGCCTTGAGTT-3'.
The kit further includes loop-mediated isothermal amplification reaction reagent and indicator.
The kit further include mycoplasma pneumoniae positive quality control standard items and/or as negative control without ribonucleic acid
The water of enzyme.
The loop-mediated isothermal amplification reaction reagent includes Bst 2.0DNA polymerases (M0537S, knob Great Britain biotechnology)
With 10mM dNTP mixtures (A610056, raw work biology).
The indicator refers to for her ten thousand Green fluorescent dyes (EvaGreen fluorescent reagents), calcein or hydroxynaphthol blue
Show agent (HNB) Metal ion indicator, preferably HNB, concentration is preferably 120 μM.
Color change observing response terminal can be passed through.EvaGreen fluorescent reagents are added in the reaction product, in ultraviolet lamp
Lower observation color change, green fluorescence are the positive, and redgreen fluorescence is feminine gender；Or after the completion of reaction be added calcein and
MnCl2, visual color variation, green is the positive, orange for feminine gender.
HNB is a kind of Metal ion indicator, and color changes with pH value of solution and changed, Mg in LAMP reaction process2+With
LAMP reaction product magnesium pyrophosphates, which combine, generates a large amount of precipitations, Mg in solution2+Concentration reduces, and pH changes, to make HNB's
Color becomes blue from purple.
HNB will not be influenced by primer dimer compared with EvaGreen fluorescent reagents and generate false positive, and will not be to anti-
Answer System forming to interfere, can directly add in the reaction system, anyway after directly visual color variation, need not beat
Reaction tube is opened, the possibility of pollution is reduced.
The outer primer F3 is 0.2 μM a concentration of, outer primer B3 is 0.2 μM a concentration of, inner primer FIP is 1.6 μM a concentration of, interior draws
A concentration of 1.6 μM of object BIP.
Above-mentioned detection mycoplasma pneumoniae kit, the positive quality control standard items are Mycoplasma pneumonia DNA, preparation method
It is culture mycoplasma pneumoniae reference culture, Mycoplasma pneumonia DNA is extracted using DNA extraction kit, to obtain positive criteria
Product.
Another object of the present invention is to provide a kind of methods detecting mycoplasma pneumoniae using ring mediated isothermal amplification, should
Method includes the following steps：
S1 extracts sample DNA；
Sample DNA and primer sets, loop-mediated isothermal amplification reaction reagent and indicator are mixed to get mixture by S2；
S3, mixture, which is placed in 64 DEG C of waters bath with thermostatic control, reacts 15min, 80 DEG C of reaction 20min inactivation Bst 2.0DNA polymerizations
Enzyme.
S4 observes color change after reaction, whether to have mycoplasma pneumoniae in judgement sample.
In step s3, mixture first of short duration centrifugation, which is placed in again in 64 DEG C of waters bath with thermostatic control, reacts 15min.
The positive effect of the present invention is that：The present invention designs 4 spies for 6 regions of mycoplasma pneumoniae P1 genes
Specific primer, amplified production have the specificity of height.The present invention reacts under constant temperature, does not need expensive temperature cycles
Instrument, it is only necessary to which the thermostatic equipments such as water-bath greatly reduce testing cost.Reaction time of the invention only needs 35min, improves
Clinically to the response time of mycoplasma pneumoniae infection, preferably patient is instructed to treat.Present invention only requires observation colors to become
Change judges reaction result, need not open reaction tube, avoid cross contamination.The kit of the present invention has high specificity, spirit
The features such as sensitivity is high, detection mycoplasma pneumoniae is convenient and efficient, cheap, is promoted the use of suitable for different medical unit.
Description of the drawings
Fig. 1 is when ring mediated isothermal amplification detects the kit of mycoplasma pneumoniae applied to clinical sample in embodiment 1
Testing result photo；
Fig. 2 is that the middle ring mediated isothermal amplification of the present invention detects the sensitivity testing result photograph of mycoplasma pneumoniae kit
Piece；
Fig. 3 is that the ring mediated isothermal amplification of the present invention detects the specific detection result photo of mycoplasma pneumoniae kit；
Wherein, 1 is mycoplasma hyorhinis, and 2 be staphylococcus aureus, and 3 be streptococcus pneumonia, and 4 be haemophilus influenzae.
Specific implementation mode
1 ring mediated isothermal amplification of embodiment detects the application of mycoplasma pneumoniae kit
1) sample collection
Infant Nasopharyngeal swabs sample is acquired, sample is placed in pathogen sample solution and preserves.
2) QIAamp DNA Mini Kit extracts kits (51306) are used to extract sample DNA：
Sample is all placed in 2mL centrifuge tubes, 13800rpm centrifuges 3min, sucks supernatant discarding, precipitation is blown and beaten
180 μ L Buffer ATL and 20 μ L Proteinase K, 56 DEG C of water-bath 3h are added in mixing.200 μ L Buffer AL are added,
200 μ L absolute ethyl alcohols are added into centrifuge tube, mix well by 70 DEG C of water-bath 10min.Spin Column are placed in
On Collection Tube, mixed solution is moved in Spin Column, 8000rpm centrifuges 1min, abandons filtrate.By 500 μ L
Buffer AW1 are added in Spin Column, and 8000rpm centrifuges 1min, abandons filtrate.Spin is added in 500 μ L Buffer AW2
In Column, 13200rpm centrifuges 3min, abandons filtrate.Spin Column are placed on Collection Tube, 13200rpm
Centrifuge 1min.Spin Column are placed on new 1.5mL Collection Tube, are added in Spin Column film centres
Enter 40 μ L ddH2O, is stored at room temperature 5min, and 8000rpm centrifuges 1min eluted dnas.
3) LAMP is detected：
By New England Biolabs Bst shown in table 1 2.0 DNA Polymerase (M0537S) kit
It is anti-that ingredient, raw work biology 10mM dNTP mixture (A610056) and Mike's woods HNB indicator (H810857) are configured to LAMP
System, the concentration of concrete component is answered to be shown in Table 1.
1 LAMP reaction systems of table form
| 10×Isothermal Amplification Buffer | 2.5μL |
| 100mM MgSO4 | 1.5μL |
| 10μM F3 | 0.5μL |
| 10μM B3 | 0.5μL |
| 10μM FIP | 4μL |
| 10μM BIP | 4μL |
| 10mM dNTP | 3.5μL |
| 2.0 archaeal dna polymerases of 8000U/mL Bst | 1μL |
| 3mM HNB | 1μL |
| ddH2O | 4.5μL |
| Template | 2μL |
| Total volume is 25 μ L |
Reaction tube is placed in 64 DEG C of thermostat water baths and reacts 15min, 80 DEG C of reaction 20min inactivate Bst2.0 polymerases.
Observe color change after reaction, Mg in LAMP reaction process2+Combined generation a large amount of heavy with LAMP reaction product magnesium pyrophosphates
It forms sediment, Mg in solution2+Concentration reduces, and pH changes, and to make the color of HNB become blue from purple, therefore purple is feminine gender,
Blue is the positive, the result is shown in Figure 1.
2 ring mediated isothermal amplification of embodiment detects mycoplasma pneumoniae kit sensitivity analysis
1) sample process：Mycoplasma pneumoniae standard is extracted using QIAamp DNA Mini Kit extracts kits (51306)
The DNA of bacterial strain is as template, specific steps：Bacterium solution is all placed in 2mL centrifuge tubes, 13800rpm centrifuges 3min, sucks
Clear liquid abandons, and precipitation is blown and beaten mixing, 180 μ L Buffer ATL and 20 μ L Proteinase K, 56 DEG C of water-bath 3h are added.Add
Enter 200 μ L Buffer AL, 70 DEG C of water-bath 10min, 200 μ L absolute ethyl alcohols are added into centrifuge tube, mix well.By Spin
Column is placed on Collection Tube, and mixed solution is moved in Spin Column, and 8000rpm centrifuges 1min, abandons filter
Liquid.500 μ L Buffer AW1 are added in Spin Column, 8000rpm centrifuges 1min, abandons filtrate.By 500 μ L Buffer
AW2 is added in Spin Column, and 13200rpm centrifuges 3min, abandons filtrate.Spin Column are placed in Collection Tube
On, 13200rpm centrifuges 1min.Spin Column are placed on new 1.5mL Collection Tube, in Spin
40 μ L ddH are added in Column film centres2O, is stored at room temperature 5min, and 8000rpm centrifuges 1min eluted dnas.
Using spectrophotometric determination DNA concentration, it is 10 to calculate DNA profiling copy number by molecular weight7Copies/ μ L,
10 times of gradient dilutions are to 101, each gradient takes 2 μ L as template, copy number 107~101copies/μL。
2) 10 0.5mL centrifuge tubes are taken, are respectively labeled as 107~101, negative control pipe；25 μ L reaction systems are configured, respectively
Following component is sequentially added in pipe, concentration is as follows：
Reaction mixture：10 times of diluted Isothermal Amplification Buffer, 6mM MgSO4、1.4mM
DNTP, 320 U/mL Bst, 2.0 archaeal dna polymerases, 120 μM of HNB.
Primer：F3 0.2μM、B3 0.2μM、FIP 1.6μM、BIP 1.6μM.
Sample-adding：10 are separately added into centrifuge tube8~101The Mycoplasma pneumonia DNA and RNase-Free of copies/ μ L
Water 2μL。
LAMP reacts：Reaction tube is placed in 64 DEG C of thermostat water baths after of short duration centrifugation and reacts 15min, 80 DEG C of reactions
20min inactivates 2.0 polymerases of Bst.Color change is observed after reaction, and purple is feminine gender, and blue is the positive.
Testing result is as shown in Figure 2, it is seen that the sensitivity of kit detection is 103copies/μL。
3 ring mediated isothermal amplification of embodiment detects the specificity analysis in real time of mycoplasma pneumoniae kit
1) sample process：Following pathogen is extracted using QIAamp DNA Mini Kit extracts kits (51306)
DNA：Mycoplasma hyorhinis, staphylococcus aureus, streptococcus pneumonia, haemophilus influenzae, specific steps：Bacterium solution is all placed in
In 2ml centrifuge tubes, 13800rpm centrifuges 3min, sucks supernatant discarding, and precipitation is blown and beaten mixing, 180 μ L Buffer are added
ATL and 20 μ L Proteinase K, 56 DEG C of water-bath 3h.200 μ L Buffer AL, 70 DEG C of water-bath 10min, to centrifuge tube are added
200 μ L absolute ethyl alcohols of middle addition, mix well.Spin Column are placed on Collection Tube, mixed solution is moved
Into Spin Column, 8000rpm centrifuges 1min, abandons filtrate.500 μ L Buffer AW1 are added in Spin Column,
8000rpm centrifuges 1min, abandons filtrate.500 μ L Buffer AW2 are added in Spin Column, 13200rpm centrifuges 3min,
Abandon filtrate.Spin Column are placed on Collection Tube, 13200rpm centrifuges 1min.Spin Column are placed in
On new 1.5mL Collection Tube, 40 μ L ddH are added in Spin Column film centres2O is stored at room temperature 5min,
8000rpm centrifuges 1min eluted dnas.
By above several pathogen DNA and Mycoplasma pneumonia DNA respectively as reaction template.
2) 6 0.5mL centrifuge tubes are taken, mycoplasma hyorhinis, staphylococcus aureus, streptococcus pneumonia, stream are respectively labeled as
Haemophilus influenza, mycoplasma pneumoniae, negative control pipe；25 μ L reaction reagents are configured, sequentially add following component in each pipe, specifically
Concentration is as follows：
Reaction mixture：10 times of diluted Isothermal Amplification Buffer, 6mM MgSO4、1.4mM
DNTP, 320 U/mL Bst, 2.0 archaeal dna polymerases, 120 μM of HNB.
Primer：F3 0.2μM、B3 0.2μM、FIP 1.6μM、BIP 1.6μM.
Sample-adding：The bloodthirsty bar of mycoplasma hyorhinis, staphylococcus aureus, streptococcus pneumonia, influenza is separately added into centrifuge tube
Bacterium, mycoplasma pneumoniae each 2 μ L of DNA and RNase-Free Water.
LAMP reacts：Reaction tube is placed in 64 DEG C of thermostat water baths after of short duration centrifugation and reacts 15min, 80 DEG C of reactions
20min inactivates 2.0 polymerases of Bst.Color change is observed after reaction, and purple is feminine gender, and blue is the positive.
Testing result as shown in figure 3, when kit detection only has mycoplasma pneumoniae as template product in blue, remaining
Respiratory pathogen and negative control are in purple, show that this kit has preferable specificity.
Sequence table
<110>Shanghai Jiaotong University Medical College subsidiary Shanghai Children's Medi
<120>Ring mediated isothermal amplification detects the kit and method of mycoplasma pneumoniae
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Mycoplasma pneumoniae (Mycoplasma pneumoniae)
<400> 1
ctggccaatc cacccaac 18
<210> 2
<211> 19
<212> DNA
<213>Mycoplasma pneumoniae (Mycoplasma pneumoniae)
<400> 2
actggaaagg gcagtacca 19
<210> 3
<211> 41
<212> DNA
<213>Mycoplasma pneumoniae (Mycoplasma pneumoniae)
<400> 3
accattgtcc tccgagtccg atgtcagggg acaccaaagt c 41
<210> 4
<211> 39
<212> DNA
<213>Mycoplasma pneumoniae (Mycoplasma pneumoniae)
<400> 4
gatctcgcca acgctcccat aaatcgtccg ccttgagtt 39
Claims (9)
1. a kind of kit of ring mediated isothermal amplification detection mycoplasma pneumoniae, which is characterized in that the kit includes primer
Group, the primer sets are by SEQ ID NO:Upstream outer primers F 3 shown in 1, SEQ ID NO:Downstream outer primer shown in 2
B3、SEQ ID NO:Upstream internal primers F IP shown in 3 and SEQ ID NO:Downstream inner primer BIP shown in 4 is formed.
2. the kit of ring mediated isothermal amplification detection mycoplasma pneumoniae as described in claim 1, which is characterized in that the examination
Agent box further includes loop-mediated isothermal amplification reaction reagent and indicator.
3. the kit of ring mediated isothermal amplification detection mycoplasma pneumoniae as claimed in claim 2, which is characterized in that the examination
Agent box further includes mycoplasma pneumoniae positive quality control standard items.
4. the kit of ring mediated isothermal amplification detection mycoplasma pneumoniae as claimed in claim 2, which is characterized in that the examination
Agent box further includes the deionized water of the deoxyribonuclease as negative control.
5. the kit of ring mediated isothermal amplification detection mycoplasma pneumoniae as claimed in claim 2, it is characterised in that the ring
Mediated isothermal amplification reaction reagent includes：Bst 2.0DNA polymerases and 10mM dNTP mixtures.
6. the kit of ring mediated isothermal amplification detection mycoplasma pneumoniae as claimed in claim 2, which is characterized in that the finger
Show agent for her ten thousand Green fluorescent dyes, calcein or hydroxynaphthol blue indicator.
7. the kit of ring mediated isothermal amplification detection mycoplasma pneumoniae as described in claim 1, which is characterized in that described outer
3 a concentration of 0.2 μM of primers F, outer primer B3 is 0.2 μM a concentration of, inner primer FIP is 1.6 μM a concentration of, inner primer BIP a concentration of 1.6
μM。
8. a kind of method detecting mycoplasma pneumoniae using ring mediated isothermal amplification, it is characterised in that include the following steps：
S1 extracts sample DNA；
Sample DNA and primer sets, loop-mediated isothermal amplification reaction reagent and indicator are mixed to get mixture by S2；
S3, mixture, which is placed in 64 DEG C of waters bath with thermostatic control, reacts 15min, and 80 DEG C of reaction 20min inactivate Bst 2.0DNA polymerases.
S4 observes color change after reaction, whether to have mycoplasma pneumoniae in judgement sample.
9. method as claimed in claim 8, which is characterized in that in step s3, first of short duration centrifugation is placed in 64 DEG C to mixture again
15min is reacted in water bath with thermostatic control.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023067510A1 (en) * | 2021-10-19 | 2023-04-27 | Life Technologies Corporation | Colorimetric detection of nucleic acids |
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| CN103276083A (en) * | 2013-05-30 | 2013-09-04 | 首都医科大学附属北京友谊医院 | Mycoplasma pneumonia detection kit |
| CN105238860A (en) * | 2015-10-15 | 2016-01-13 | 中国人民解放军疾病预防控制所 | LAMP (loop-mediated isothermal amplification) kit for detection of mycoplasma pneumoniae and special LAMP primer for detection of mycoplasma pneumoniae |
| CN106399542A (en) * | 2016-10-26 | 2017-02-15 | 沈阳优宁生物科技有限公司 | Rolling circle constant-temperature amplification quick detection primer and kit for mycoplasma pneumoniae |
| CN107236798A (en) * | 2017-06-08 | 2017-10-10 | 天津医科大学 | The method that loop-mediated isothermal amplification technique detects mycoplasma pneumoniae |
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2018
- 2018-07-02 CN CN201810704561.8A patent/CN108642196A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618655A (en) * | 2012-04-16 | 2012-08-01 | 中国疾病预防控制中心传染病预防控制所 | Loop-mediated isotherm amplification (LAMP) kit for detecting mycoplasma pneumoniae (Mp) |
| CN103276083A (en) * | 2013-05-30 | 2013-09-04 | 首都医科大学附属北京友谊医院 | Mycoplasma pneumonia detection kit |
| CN105238860A (en) * | 2015-10-15 | 2016-01-13 | 中国人民解放军疾病预防控制所 | LAMP (loop-mediated isothermal amplification) kit for detection of mycoplasma pneumoniae and special LAMP primer for detection of mycoplasma pneumoniae |
| CN106399542A (en) * | 2016-10-26 | 2017-02-15 | 沈阳优宁生物科技有限公司 | Rolling circle constant-temperature amplification quick detection primer and kit for mycoplasma pneumoniae |
| CN107236798A (en) * | 2017-06-08 | 2017-10-10 | 天津医科大学 | The method that loop-mediated isothermal amplification technique detects mycoplasma pneumoniae |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023067510A1 (en) * | 2021-10-19 | 2023-04-27 | Life Technologies Corporation | Colorimetric detection of nucleic acids |
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