CN104388558B - The molecular beacon probe of a kind of quick detection streptococcus agalactiae and detection method - Google Patents
The molecular beacon probe of a kind of quick detection streptococcus agalactiae and detection method Download PDFInfo
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- CN104388558B CN104388558B CN201410635596.2A CN201410635596A CN104388558B CN 104388558 B CN104388558 B CN 104388558B CN 201410635596 A CN201410635596 A CN 201410635596A CN 104388558 B CN104388558 B CN 104388558B
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Abstract
The invention discloses the molecular beacon probe of a kind of quick detection streptococcus agalactiae, it is characterised in that the base sequence of described molecular beacon probe is: Beacon SAG:5 ' FAM CACCATTCTGCTCCGAAGAGAAAGCTGGTG DABCAL 3 '；5 ' end FAM labellings of described probe, 3 ' end DABCAL labellings, fluorophor excitation wavelength 495nm, detects wavelength 520nm.The molecular beacon probe signal intensity of the present invention is high, and specificity is high, can detect streptococcus agalactiae effectively and quickly.The invention also discloses test kit and the detection method of a kind of quick detection streptococcus agalactiae.
Description
Technical field
The invention belongs to biology field, particularly to a kind of molecular beacon probe and test kit, by using fluorescent in situ
The means of hybridization, streptococcus agalactiae a small amount of in detection sample.
Background technology
Streptococcus agalactiae is also known as B group streptococcus (Group B Streptococcus, GBS), is neonate and female reproductive system
The significant bacterial of togetherness dye.The especially infection of non-neonate, is life-threatening major reason, and its complication includes deteriorated blood
Disease, pneumonia and meningitis etc..In the intravaginal of adult women, have about 15% can detect streptococcus agalactiae, this kind of women
The probability of this bacterium of infection of newborn of childbirth can be higher.
Neonate Streptococcus agalactiae infections is divided into early onset to infect and late-onset infects two kinds.Early onset infect be usually infect after one
Morbidity in week, the mortality rate infected at U.S.'s neonate early onset is 4%-6%；Hair style infected and was more common in be born latter 7 days～March evening
Term infant, mainly shows as meningitis, often in invisible morbidity.Clinical manifestation has heating, lethargy, intracranial hypertension etc., such as companion
Send out septicemia then prognosis poor.Most infection is all because the streptococcus agalactiae that parent reproductive system carries, via the fetal membrane ruptured
Enter amniotic fluid, then infect fetus.Antibacterial infects respiratory tract can cause pneumonia, enters blood system and can cause septicemia, meninges
Inflammation and osteomyelitis etc..
Detection streptococcus agalactiae most common method is CAMP method the most clinically: on blood plate, B group streptococcus can produce CAMP
The factor, can promote staphylococcus aureus hemolysin activity with the standardized bar of S. aureus culture on blood agar plate
Straight line, then draws a line streptococcus culture, and the line with S. aureus culture is mutually perpendicular to, but does not connect
Touching, the two separates bacterium scribe line pitch l0～20mm at a distance of 3-5mm, each streptococcus.Knot is observed after cultivating 24h in putting 37 DEG C of incubators
Really.The haemolysis district of arrow sample occurs for positive at two line intersections.The method has simply, the advantage of low cost, and shortcoming is
Sensitivity is relatively low, and the detection time is longer.In sample, bacteria containing amount tends not to draw positive findings time few, and cannot distinguish between and be
Streptococcus agalactiae or other B group streptococcus.Utilize PCR method can make specific nucleotide sequence copy number geometry at short notice
Progression increases, and has the highest sensitivity and specificity, but shortcoming is false positive rate height, on the other hand, has past in patient's secretions
Toward the composition containing suppression PCR reaction, this can cause again false-negative appearance, and the shortcoming of these two aspects limits PCR detection
Clinical practice.As can be seen here, streptococcus agalactiae clinical diagnosis, it is badly in need of more succinct, sensitive detection method.
Molecular beacon probe (Molecular beacon probe), has highly sensitive, high specificity, only and target sequence is miscellaneous
Hand in and just can send the advantages such as fluorescence, be applied to fluorescence in situ hybridization.The present invention by a large amount of streptococcus agalactiaes and other
The 16S rRNA sequence of strains of streptococcus is compared, and selects the specific sequence of streptococcus agalactiae, design, synthetic molecules beacon
Probe, sets up stable molecular beacon in situ hybridization reaction system, overcomes the problems of streptococcus agalactiae Present clinical detection,
For Streptococcus agalactiae infections diagnosis, prevent and control to provide new detection method.
Summary of the invention
It is an object of the invention to provide a kind of molecular beacon probe, by the means of fluorescence in situ hybridization, set up a kind of quickly,
Sensitive, the specifically method of streptococcus agalactiae in detection sample.
The molecular beacon probe of a kind of quick detection streptococcus agalactiae, it is characterised in that the base sequence of described molecular beacon probe
For:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '；
5 ' end FAM labellings of described probe, 3 ' end DABCAL labellings, fluorophor excitation wavelength 495nm, detects wavelength
520nm。
Further, described molecular beacon concentration is 10ng/ μ L.
Present invention also offers the test kit of a kind of quick detection streptococcus agalactiae, it is characterised in that described test kit includes:
(1) lysate: 4% sodium hydroxide
(2) hybridization solution: 10% (w/v) dextran sulfate, 10mM NaCl, 20% (v/v) Methanamide, 0.1% (w/v) burnt phosphorus
Acid sodium, 0.2% (w/v) polyvinyl pyrrolidone, 0.2% (w/v) poly-deer sugar, 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
It is classified as:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '；
(3) stop buffer: 1% dilute sulfuric acid
(4) cleaning mixture: 5mM Tris, 15mM NaCl, 0.1% (v/v) Triton X-100, pH value is 10.
The present invention finally provides a kind of method that mentioned reagent box quickly detects streptococcus agalactiae, it is characterised in that include walking as follows
Rapid:
(1) draw 10 μ L sample and drop on microscope slide, natural air drying；
(2) on air-dried sample, add 10 μ L lysates, after its natural air drying, immerse in dehydrated alcohol, soak 5 points
Clock；
(3) 52 DEG C hybridize 10 minutes；
(4) liquid invades bubble 1 minute, terminates reaction；
(5) after dropping mountant, with fluorescence microscopy, with the pan of 20 × object lens with count, with 60 or 100 × object lens
Observe ne ar.
Technical key point or principle: fluorescence in situ hybridization (Flourescence in situ Hybridization, FISH) is
A kind of application is marked with the probe of fluorescent material, detects cell or tissue internal specific DNA or RNA by the method for hybridization
Method；Molecular beacon probe is the probe that one has uniqueness " hair clip " space structure, when not being combined with target sequence, point
Sub-beacon, in " hair clip " structure, has a ring sequence (loop) and a stem sequence (stem), and its medium ring sequence is mutual with target site
The base sequence mended, and stem sequence is the complementary series unrelated with target site；Be marked with respectively at the two ends of probe fluorophor and
Fluorescent quenching group, when probe is in hairpin structure, fluorophor and quencher are adjacent, produce resonance energy transfer effect,
Fluorophor is quenched, it is impossible to produce fluorescence signal, and when probe is combined with target site, hairpin structure is opened, glimmering
Light group and quencher separately, produce fluorescence signal, this fluorescence signal can be detected by fluorescence microscope.
The present invention, by the multiple streptococcus agalactiae of comparison and other streptococcic 16S rRNA sequence, filters out 1 agalactia hammer
The distinctive target sequence of bacterium.According to this target sequence, synthetic molecular beacon probe, its base composition is:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '
5 ' end FAM labellings of probe, 3 ' end DABCAL labellings, fluorophor excitation wavelength 495nm, detects wavelength 520nm.
The present invention passes through great many of experiments, determines that the optimum temperature of fluorescence in situ hybridization is 52 DEG C, and Methanamide optium concentration is 20%,
Molecular beacon optium concentration is 10ng/ μ L.
The fluorescent labeling that molecular beacon probe 5 ' of the present invention is held, includes but not limited to FITC, FAM or Cy3 etc., 3 ' ends
Fluorescent quenching labelling, include but not limited to that DABCYL, BDH or TANRA etc., fluorophor or fluorescent quenching group can
To be added according to prior art.
The sampling range that the molecular beacon probe of the present invention can be used in detection is extensive, includes but not limited to, expectorant, throat swab, stomach
Irrigating solution, bronchial perfusate, biological tissue, attraction, begma, body fluid (spinal cord, ascites pleural fluid, pericardial fluid etc.), blood
Liquid, pus, bone marrow, urine, tissue slice, food sample, sample from soil, air and water, and their training
Support thing.These samples are after respective handling, as long as being essentially, holding cellular morphology is complete and target nucleic acid is not destroyed,
Use the molecular beacon probe detection of the present invention.The processing method of these samples is that those skilled in the art are grasped, such as:
Expectorant: sputum smear method；
Pus: with sputum smear method；
Lesion tissue: row smear again after first grinding with tissue grinder；
Urine: stay full dose nocturia, after standing 4～5h, abandons supernatant, takes sediment fraction urine 10ml, 3000rpm, is centrifuged
30min, taking precipitate smear；
Breast, ascites specimen: with reference to urine smear method；
Cerebrospinal fluid: cerebrospinal fluid is collected by sterile working, places refrigerator or room temperature 24h, smear after thin film is formed.Also can be by brain ridge
Liquid centrifugation, 3000rpm, centrifugal 30min, abandon supernatant, taking precipitate smear.
The molecular beacon probe signal intensity of the present invention is high, and specificity is high, can detect streptococcus agalactiae effectively and quickly.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but the following example is merely to illustrate this
Bright, and should not be taken as limiting the scope of the invention.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or instrument unreceipted production firm person, be can by city available from conventional products.
Embodiment 1: molecular beacon probe and the design of oligonucleotide sequences and synthesis
Select the target sequence that can specifically detect streptococcus agalactiae, design and the molecule of its complete complementary on this section of target sequence
Beacon probe:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '
This molecular beacon is by the distinguished sequence of the neck ring structure of base composition, wherein 5, and end uses FAM labelling, 3, end DABCTL
Labelling, fluorophor requires excitation wavelength 495nm, detects wavelength 520nm；Engineer's synthetic molecules beacon and complete with it
Complementary oligonucleotide (5 '-AGGTCCGGGTTCTCTCGGAT-3 ').By molecular beacon and oligonucleotide being done heat change
Linearity curve is tested, and determines that the optimal reaction temperature of fluorescence in situ hybridization is 52 DEG C, and the optium concentration of deionized formamide is 20%.
Embodiment 2: use molecular beacon probe FISH to detect antibacterial reference culture.
Streptococcus agalactiae and other antibacterial reference cultures, purchased from ATCC, detect 5 kinds of streptococcus agalactiaes and 12 kinds of other streptococcus altogether.
Detection kit includes:
(1) lysate: 4% sodium hydroxide
(2) hybridization solution: 10% (w/v) dextran sulfate, 10mM NaCl, 20% (v/v) Methanamide, 0.1% (w/v) burnt phosphorus
Acid sodium, 0.2% (w/v) polyvinyl pyrrolidone, 0.2% (w/v) poly-deer sugar, 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
It is classified as:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '；
(3) stop buffer: 1% dilute sulfuric acid
(4) cleaning mixture: 5mM Tris, 15mM NaCl, 0.1% (v/v) Triton X-100, pH value is 10.
Detection method:
(1) draw 10 μ L culture sample and drop on microscope slide, natural air drying；
(2) on air-dried sample, add 10 μ L lysates, after its natural air drying, immerse in dehydrated alcohol, soak 5 points
Clock；
(3) on sample, add 10 μ L hybridization solutions, be placed in hybrid heater 52 DEG C and hybridize 10 minutes；
(4) invade bubble 1 minute with stop buffer, terminate reaction；
(5) after dropping mountant, with fluorescence microscopy, with the pan of 20 × object lens with count, with 60 or 100 × object lens
Observe ne ar.In dark background, streptococcus agalactiae sends green fluorescence.
Result decision method:
Streptococcus agalactiae feminine gender (-): 50 different visuals field of Continuous Observation, do not find antibacterial.
Streptococcus agalactiae positive (report bacterium number): the visual field, 1-9 bar/50.
Streptococcus agalactiae is positive (1+): the visual field, 10-99 bar/50.
Streptococcus agalactiae is positive (2+): the every visual field of 1-9 bar.
Streptococcus agalactiae is positive (3+): the every visual field of 10-99 bar.
Streptococcus agalactiae positive (+): > 100 every visuals field.
Table one: use streptococcus agalactiae molecular beacon probe FISH to detect antibacterial reference culture
Analysis of test results: 5 kinds of streptococcus agalactiae bacterial strain testing results are all positive, and 12 kinds of other antibacterials, testing result is all
Feminine gender, illustrates that this detection method detection streptococcus agalactiae has good specificity.
SEQUENCE LISTING
<110>
Side, Hua Cheng
<120>
The molecular beacon probe of a kind of quick detection streptococcus agalactiae and detection method
<130>
<160>
1
<170>
PatentIn version 3.3
<210>
1
<211>
30
<212>
DNA
<213>
Artificial sequence
<400>
1
caccattctg
ctccgaagag
aaagctggtg
30
Claims (2)
1. the molecular beacon probe of a quick detection streptococcus agalactiae, it is characterised in that the base sequence of described molecular beacon probe is:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '；
5 ' end FAM labellings of described probe, 3 ' end DABCAL labellings, fluorophor excitation wavelength 495nm, detects wavelength
520nm。
2. the test kit of a quick detection streptococcus agalactiae, it is characterised in that described test kit includes:
(1) lysate: 4% sodium hydroxide；
(2) hybridization solution: 10%w/v dextran sulfate, 10mM NaCl, 20%v/v Methanamide, 0.1%w/v sodium pyrophosphate,
0.2%w/v polyvinyl pyrrolidone, 0.2%w/v poly-deer sugar, 5mM Na2EDTA, 0.1%v/vTritonX-100,50mM
PH value is the Tris/HCl of 7.5,10ng/ μ L molecular beacon probe, and the base sequence of described molecular beacon probe is:
Beacon SAG:5 '-FAM-CACCATTCTGCTCCGAAGAGAAAGCTGGTG-DABCAL-3 '；
(3) stop buffer: 1% dilute sulfuric acid；
(4) cleaning mixture: 5mM Tris, 15mM NaCl, 0.1%v/v Triton X-100, pH value is 10.
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| CN108342499A (en) * | 2018-04-26 | 2018-07-31 | 暨南大学 | A pair of while quickly detection Streptococcusagalactiae and Streptococcus iniae primer and its application |
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| CN106546724A (en) * | 2015-09-23 | 2017-03-29 | 中国医学科学院药用植物研究所 | A kind of new method of molecular beacon probe quick detection ochratoxin A |
| CN105695599A (en) * | 2016-03-25 | 2016-06-22 | 苏州达麦迪生物医学科技有限公司 | Molecular beacon probe capable of rapidly detecting rifampicin drug-resistant mycobacterium tuberculosis and kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108342499A (en) * | 2018-04-26 | 2018-07-31 | 暨南大学 | A pair of while quickly detection Streptococcusagalactiae and Streptococcus iniae primer and its application |
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