CN104388557A - Molecular beacon probe and detection method for rapidly detecting pyogenic streptococcus - Google Patents

Abstract

The invention discloses a molecular beacon probe for rapidly detecting pyogenic streptococcus. The molecular beacon probe is characterized in that the base sequence of the molecular beacon probe is BeaconSPY: 5'-CY3-CATTG TACGCCCATAATTCGGAC CAATG-BHQ1-3', wherein the 5' terminal of the probe is marked with Cy3, the 3' terminal of the probe is marked with BHQ1, and the excitation wavelength of fluorophore is 552nm and the detection wavelength is 570nm. The molecular beacon probe has high signal intensity and high specificity and can be used for effectively and rapidly detecting pyogenic streptococcus. The invention also discloses a kit and a detection method, which are used for rapidly detecting pyogenic streptococcus.

Description

A kind of molecular beacon probe of rapid detection micrococcus scarlatinae and detection method

Technical field

The invention belongs to biology field, particularly a kind of molecular beacon probe and test kit, by using the means of fluorescence in situ hybridization, detecting micrococcus scarlatinae a small amount of in sample.

Background technology

Micrococcus scarlatinae, also known as A group B streptococcus B (GAS), is one of most important cause of disease in human bacterial infections.Micrococcus scarlatinae can be invaded and any position of human body, but is modal primary infection position with the upper respiratory tract, secondly skin soft-tissue infection.Micrococcus scarlatinae can cause purulent disease and apyetous complication two class disease.The apyetous complication indirectly caused by micrococcus scarlatinae and immunological disease: rheumatic fever and acute glomerulonephritis.Micrococcus scarlatinae hypotype is many, various without cross immunity, therefore normal repeated infection.Severe infections in recent years caused by micrococcus scarlatinae, the growth of aggressive S. pyogenes infection sickness rate and caused serious consequence thereof, also result in the concern that people are larger to this bacterial infection.

The method detecting micrococcus scarlatinae the most frequently used clinically is at present Smear detection, takes different samples according to various disease.As the fester of wound, the cotton of the focus such as throat, nasal cavity is wiped, and gets blood etc. during septicemia.Serum is got when detecting antibody.Direct smear microscopy fester sample can direct smear, microscopy after gram's staining.During fester sample Microscopic observation, bacterium Chang Chengshuan or exist with single form, instead of in typical chain.The advantage that the method has simply, quick, cost is low, shortcoming is that sensitivity is low, often can not draw positive findings in phlegm or when bacteria containing amount is few in other sample.

The result of isolated culture is relatively reliable, and specificity is high, is the gold standard of current diagnosis S. pyogenes infection.But micrococcus scarlatinae can not be cultivated in plain agar flat board and broth culture, and needing to add blood in the medium can grow, and sometimes also needs to add multiple somatomedin in the medium, and needs 3-5 days just can see detected result.As can be seen here, the clinical diagnosis of micrococcus scarlatinae, is badly in need of more succinct, sensitive detection method.

Molecular beacon probe (Molecular beacon probe), has highly sensitive, high specificity, only has to have gone up with target sequence hybridization and just can send the advantages such as fluorescence, be applied to fluorescence in situ hybridization.The present invention is by comparing to the 16S rRNA sequence of a large amount of mycobacterium tuberculosis and micrococcus scarlatinae bacterial strain, select the specific sequence of micrococcus scarlatinae, design, synthetic molecules beacon probe, set up stable molecular beacon in situ hybridization reaction system, overcome the problems that micrococcus scarlatinae Present clinical detects.

Summary of the invention

The object of the present invention is to provide a kind of molecular beacon probe, by the means of fluorescence in situ hybridization, set up a kind of quick, sensitive, method of detecting micrococcus scarlatinae in sample specifically.

A molecular beacon probe for rapid detection micrococcus scarlatinae, is characterized in that, the base sequence of described molecular beacon probe is:

Beacon SPY：5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’；

5 ' end Cy3 of described probe marks, 3 ' end BHQ1 mark, fluorophor excitation wavelength 552nm, determined wavelength 570nm.

Further, described molecular beacon concentration is 10ng/ μ L.

Present invention also offers a kind of test kit of rapid detection micrococcus scarlatinae, it is characterized in that, described test kit comprises:

(1) lysate: 4% sodium hydroxide

(2) hybridization solution: 10% (w/v) T 500,10mM NaCl, 20% (v/v) methane amide, 0.1% (w/v) trisodium phosphate, 0.2% (w/v) Polyvinylpyrolidone (PVP), the poly-deer sugar of 0.2% (w/v), 5mM Na2EDTA, 0.1% (v/v) TritonX-100,50mM Tris/HCl (pH 7.5), 10ng/ μ L molecular beacon probe, the base sequence of described molecular beacon probe is:

Beacon SPY：5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’；

(3) stop buffer: 1% dilute sulphuric acid;

(4) washings: 5mM Tris, 15mM NaCl, 0.1% (v/v) Triton X-100, pH value is 10.

The present invention finally provides a kind of method of mentioned reagent box rapid detection micrococcus scarlatinae, it is characterized in that comprising the steps:

(1) 10 μ L sample drops are drawn on slide glass, natural air drying;

(2) on air-dry sample, add 10 μ L lysates, after its natural air drying, immerse in dehydrated alcohol, soak 5 minutes;

Hybridize 10 minutes for (3) 52 DEG C;

(4) liquid invades bubble 1 minute, termination reaction;

(5) after dripping mountant, with fluorescence microscopy, with the pan of 20 × object lens and counting, ne ar is observed with 60 or 100 × object lens.

Technical essential of the present invention or principle: fluorescence in situ hybridization (Flourescence in situ Hybridization, FISH) be the probe that a kind of application is marked with fluorescent substance, detected the method for cell or tissue internal specific DNA or RNA by the method for hybridization; Molecular beacon probe is the probe that one has uniqueness " hair clip " space structure, not with target sequence in conjunction with time, molecular beacon is in " hair clip " structure, there are a ring sequence (loop) and a stem sequence (stem), wherein ring sequence is the base sequence with target site complementation, and stem sequence is the complementary sequence irrelevant with target site; Fluorophor and quenching of fluorescence group is marked with respectively at the two ends of probe, when probe is in hairpin structure, fluorophor is adjacent with quencher, and generate energy resonance transfer effect, makes fluorophor by quencher, fluorescent signal can not be produced, and when probe and target site in conjunction with time, hairpin structure is opened, and fluorophor and quencher are separately, produce fluorescent signal, this fluorescent signal can be detected by fluorescent microscope.

The present invention, by the multiple micrococcus scarlatinae of comparison and other streptococcic 16S rRNA sequence, filters out 1 distinctive target sequence of micrococcus scarlatinae.According to this target sequence, synthetic molecular beacon probe, its based composition is:

Beacon SPY：5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’；

5 ' the end Cy3 mark of probe, 3 ' end BHQ1 mark, fluorophor excitation wavelength 552nm, determined wavelength 570nm.

The present invention is by great many of experiments, and determine that the optimum temps of fluorescence in situ hybridization is 52 DEG C, methane amide optimum concn is 20%, and molecular beacon optimum concn is 10ng/ μ L.

The fluorescent mark that molecular beacon probe 5 ' of the present invention is held, include but not limited to FITC, FAM or Cy3 etc., 3 ' the quenching of fluorescence mark held, include but not limited to DABCYL, BDH, BHQ1 or TANRA etc., fluorophor or quenching of fluorescence group can add according to prior art.

The sample range that molecular beacon probe of the present invention can be used in detecting is extensive, include but not limited to, phlegm, throat swab, gastric lavage liquid, bronchial perfusate, biological tissue, attraction, begma, body fluid (spinal cord, ascites pleural fluid, pericardial fluid etc.), blood, fester, marrow, urine, tissue slice, food sample, sample from soil, empty G&W, and their culture.These samples, after respective handling, as long as be keep the complete and target nucleic acid of cellular form not to be destroyed in principle, all can use molecular beacon probe of the present invention to detect.The treatment process of these samples is that those skilled in the art grasped, such as:

Phlegm: sputum smear method;

Fester: with sputum smear method;

Lesion tissue: row smear again after first grinding with tissue grinder;

Urine: stay full dose enuresis nocturna, after leaving standstill 4 ~ 5h, abandons supernatant liquor, gets sediment fraction urine 10ml, 3000rpm, centrifugal 30min, taking precipitate smear;

Chest, ascites sample: with reference to urine smear method;

Cerebrospinal fluid: cerebrospinal fluid is collected in aseptic technique, places refrigerator or room temperature 24h, smear after film is formed.Also can by cerebrospinal fluid centrifugation, 3000rpm, centrifugal 30min, abandons supernatant liquor, taking precipitate smear.

Molecular beacon probe strength of signal of the present invention is high, and specificity is high, can detect micrococcus scarlatinae effectively and quickly.

Embodiment

Below in conjunction with embodiment, embodiment of the present invention are described in detail, but the following example is only for illustration of the present invention, and should be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

Embodiment 1: the Design and synthesis of molecular beacon probe and oligonucleotide sequences

Select the target sequence that can detect micrococcus scarlatinae specifically, the molecular beacon probe of design and its complete complementary on this section of target sequence:

Beacon SPY：(5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’)；

This molecular beacon is by the distinguished sequence of the neck ring structure of based composition, 5 ' the end Cy3 mark of its middle probe, 3 ' end BHQ1 mark, fluorophor excitation wavelength 552nm, determined wavelength 570nm; Engineer's synthetic molecules beacon and with the oligonucleotide of its complete complementary (5 '-GTCCGGAATTACTGGGCGTA-3 ').By doing thermal denaturation curve experiment to molecular beacon and oligonucleotide, determine that the optimal reaction temperature of fluorescence in situ hybridization is 52 DEG C, the optimum concn of deionized formamide is 20%.

Embodiment 2: use branch beacon probe FISH detection of streptococcus reference culture.

Suis reference culture, purchased from ATCC, detects 5 kinds of micrococcus scarlatinaes and 12 kinds of other suis altogether.

Detection kit comprises:

(1) lysate: 4% sodium hydroxide

(2) hybridization solution: 10% (w/v) T 500,10mM NaCl, 20% (v/v) methane amide, 0.1% (w/v) trisodium phosphate, 0.2% (w/v) Polyvinylpyrolidone (PVP), the poly-deer sugar of 0.2% (w/v), 5mM Na2EDTA, 0.1% (v/v) TritonX-100,50mM Tris/HCl (pH 7.5), 10ng/ μ L molecular beacon probe, the base sequence of described molecular beacon probe is:

Beacon SPY：5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’；

(3) stop buffer: 1% dilute sulphuric acid

(4) washings: 5mM Tris, 15mM NaCl, 0.1% (v/v) Triton X-100, pH value is 10.

Detection method:

(1) drawing 10 μ L Sputum samples drops on slide glass, natural air drying;

(2) on air-dry sample, add 10 μ L lysates, after its natural air drying, immerse in dehydrated alcohol, soak 5 minutes;

(3) on sample, add 10 μ L hybridization solutions, be placed in hybrid heater 52 DEG C hybridization 10 minutes;

(4) bubble is invaded 1 minute, termination reaction with stop buffer;

(5) after dripping mountant, with fluorescence microscopy, with the pan of 20 × object lens and counting, ne ar is observed with 60 or 100 × object lens.In dark background, micrococcus scarlatinae sends red fluorescence.

Result decision method:

Micrococcus scarlatinae feminine gender (-): Continuous Observation 50 different visuals field, do not find bacterium.

The micrococcus scarlatinae positive (report bacterium number): the visual field, 1-9 bar/50.

The micrococcus scarlatinae positive (1+): the visual field, 10-99 bar/50.

The micrococcus scarlatinae positive (2+): the every visual field of 1-9 bar.

The micrococcus scarlatinae positive (3+): the every visual field of 10-99 bar.

The micrococcus scarlatinae positive (+): the every visual field of >100 bar.

Detected result is as shown in table 1.

Table 1 uses micrococcus scarlatinae molecular beacon probe FISH bacterial detection reference culture

Analysis of test results: 5 kinds of micrococcus scarlatinae detected results are positive entirely, 12 kinds of other bacteriums, and detected result is negative entirely, illustrates that this detection method detects micrococcus scarlatinae and has good specificity.

SEQUENCE LISTING

 

<110> flood, slowly

 

The molecular beacon probe of a <120> rapid detection micrococcus scarlatinae and detection method

 

<130>

 

<160> 1

 

<170> PatentIn version 3.3

 

<210> 1

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 1

cattgtacgc ccagtaattc cggaccaatg 30

 

Claims (4)

1. a molecular beacon probe for rapid detection micrococcus scarlatinae, is characterized in that, the base sequence of described molecular beacon probe is:

Beacon SPY：5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’；

5 ' end Cy3 of described probe marks, 3 ' end BHQ1 mark, fluorophor excitation wavelength 552nm, determined wavelength 570nm.

2. molecular beacon probe as claimed in claim 1, it is characterized in that, described molecular beacon concentration is 10ng/ μ L.

3. a test kit for rapid detection micrococcus scarlatinae, is characterized in that, described test kit comprises:

(1) lysate: 4% sodium hydroxide

(2) hybridization solution: 10% (w/v) T 500,10mM NaCl, 20% (v/v) methane amide, 0.1% (w/v) trisodium phosphate, 0.2% (w/v) Polyvinylpyrolidone (PVP), the poly-deer sugar of 0.2% (w/v), 5mM Na2EDTA, 0.1% (v/v) TritonX-100,50mM Tris/HCl (pH 7.5), 10ng/ μ L molecular beacon probe, the base sequence of described molecular beacon probe is:

Beacon SPY：5’-CY3-CATTGTACGCCCAGTAATTCCGGACCAATG–BHQ1-3’；

(3) stop buffer: 1% dilute sulphuric acid;

(4) washings: 5mM Tris, 15mM NaCl, 0.1% (v/v) Triton X-100, pH value is 10.

4. utilize a method for test kit rapid detection micrococcus scarlatinae described in claim 3, it is characterized in that comprising the steps:

(1) 10 μ L sample drops are drawn on slide glass, natural air drying;

(2) on air-dry sample, add 10 μ L lysates, after its natural air drying, immerse in dehydrated alcohol, soak 5 minutes;

(3) on sample, add 10 μ L hybridization solutions, be placed in hybrid heater 52 DEG C hybridization 10 minutes;

(4) bubble is invaded 1 minute, termination reaction with stop buffer;

(5) after dripping mountant, with fluorescence microscopy, with the pan of 20 × object lens and counting, ne ar is observed with 60 or 100 × object lens.