CN110964841A - Molecular beacon probe and kit for detecting septicemia and detection method thereof - Google Patents

Abstract

A molecular beacon probe, a kit and a detection method for detecting septicemia are characterized in that the base sequence of the molecular beacon probe is as follows: 5'-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-3'; the molecular beacon probe has high signal intensity and high specificity, and can effectively and quickly detect septicemia.

Description

Molecular beacon probe and kit for detecting septicemia and detection method thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a molecular beacon probe and a kit, which can detect a small amount of pathogenic bacteria contained in a sample by using a fluorescence in-situ hybridization method, namely a molecular beacon probe, a kit and a detection method thereof for detecting septicemia.

Background

Septicemia (septicemia) refers to acute systemic infection caused by invasion of pathogenic bacteria or pathogenic bacteria into blood circulation, growth and reproduction in blood, and toxin production, and is easy to cause morbidity and mortality in newborn infants. The world health organization predicts that 100 million newborns die each year from neonatal sepsis. The main pathogenic bacteria of septicemia are reported differently in different regions, and common pathogenic bacteria include Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis), Klebsiella pneumoniae (Klebsiella pneumoniae), streptococcus pneumoniae (streptococcus pneumoniae), Escherichia coli (Escherichia coli), and the like. Timely diagnosis and early treatment are key to reducing complications and mortality. However, accurate diagnosis of neonatal sepsis is difficult because of the lack of specificity in symptoms and manifestations.

At present, the clinical blood culture method is considered as the 'gold standard' for detecting septicemia, but the detection result can be obtained after 48-72 hours generally, the sensitivity is low, and false positive or false negative can occur. In recent years, researchers at home and abroad use the PCR technology for identification, the sensitivity and the specificity are high, but a bacteria detection system based on the PCR is easy to be polluted and interfered, and false positive is generated.

The molecular beacon probe (molecular beacon probe) has the advantages of high sensitivity, strong specificity, fluorescence emission only when a target sequence is hybridized and the like, and is applied to fluorescence in situ hybridization.

Disclosure of Invention

The invention aims to provide a molecular beacon probe for quickly, sensitively and specifically detecting septicemia in a sample.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a molecular beacon probe for detecting septicemia is characterized in that the base sequence of the molecular beacon probe is as follows: 5'-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-3'; (SEQ ID NO: 1).

The 5' end of the molecular beacon probe is provided with a fluorescent label, and the fluorescent label comprises but is not limited to FITC, FAM or Cy 3; the 3' end is provided with a fluorescence quenching mark, the fluorescence quenching mark comprises DABCYL, BDH, BHQ1 or TAMRA and the like, and a fluorescent group or a fluorescence quenching group can be added according to the prior art; the fluorescent group emission wavelength is 552nm, and the detection wavelength is 570 nm.

Preferably, the fluorescence label of the 5 'end of the molecular beacon probe is Cy3, the fluorescence quenching of the 3' end is BHQ1, and the base sequence of the corresponding molecular beacon probe is 5 '-Cy 3-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-BHQ 1-3'.

The invention also provides a kit containing the molecular beacon probe, and the kit can detect septicemia.

Preferably, the kit further comprises lysis solution, hybridization solution, stop solution and washing solution.

Further, the lysis solution is 4% (w/v) sodium hydroxide, and the hybridization solution is: 8-12% (w/v) dextran sulfate, 8-12 mM NaCl, 16-24% (v/v) formamide, 0.08-0.12% (w/v) sodium pyrophosphate, 0.16-0.24% (w/v) polyvinylpyrrolidone, 0.16-0.24% (w/v) polyglucoside, 4-6 mM Na₂EDTA, 0.08-0.12% (v/v) Triton X-100, 40-60 mM Tris/HCl (pH 7.5); the stop solution is 0.08-0.12% (v/v) dilute sulfuric acid; the washing solution is 4-6 mM Tris, 14-16 mM NaCl, 0.08-0.12% (v/v) Triton X-100, and the pH value is 9.5-10.5.

Further, the concentration of the molecular beacon probe in the kit is 5-20 ng/mu l.

The invention also provides a method for rapidly detecting septicemia by using the kit, which is characterized by comprising the following steps:

(1) dripping 10 mul of sample on a glass slide, and naturally drying;

(2) adding 10 μ l of lysis solution into the air dried sample, air drying, soaking in anhydrous ethanol for 5 min;

(3) adding 8-12 mul of hybridization solution and a molecular beacon probe into a sample, and placing the sample in a hybridization furnace for hybridization for 8-12 minutes at 52 ℃;

(4) soaking for 1 minute by using a stop solution, and stopping the reaction;

(5) and (5) washing with a washing solution.

(6) After addition of the encapsulated tablets, they were examined by fluorescence microscopy, scanned and counted with a 20X objective and observed for bacterial morphology with a 60 or 100X objective.

The technical key points or principles of the invention are as follows: fluorescence In Situ Hybridization (FISH) is a method for detecting specific DNA or RNA in cells or tissues by hybridization using a probe labeled with a fluorescent substance; the molecular beacon probe is a probe with a unique hairpin space structure, and when the molecular beacon is not combined with a specific target sequence of septicemia pathogenic bacteria, the molecular beacon is in the hairpin structure and has a loop sequence and a stem sequence (stem), wherein the loop sequence is a base sequence complementary with a target site, and the stem sequence is a complementary sequence unrelated to the target site; the probe is characterized in that a fluorescent group and a fluorescence quenching group are respectively marked at two ends of the probe, when the probe is in a hairpin structure, the fluorescent group and the quenching group are adjacent to generate an energy resonance transfer effect, so that the fluorescent group is quenched and cannot generate a fluorescent signal, when the probe is combined with a specific target sequence of septicemia pathogenic bacteria, the hairpin structure is opened, the fluorescent group and the quenching group are separated to generate the fluorescent signal, and the fluorescent signal can be detected through a fluorescence microscope. In other bacteria, without this specific target sequence, the probe hairpin structure cannot open and cannot generate a fluorescent signal.

The invention compares the gene sequences of the most common pathogenic bacteria in five septicemia of staphylococcus aureus, staphylococcus epidermidis, escherichia coli, streptococcus pneumoniae and klebsiella pneumoniae, selects specific gene sequences commonly owned by the five bacteria as detection targets, namely the five bacteria are completely the same and different sequences from other bacteria, designs and synthesizes a molecular Beacon probe Beacon SEP aiming at the target sites, and the basic group composition of the Beacon SEP is as follows: beacon SEP:5 '-Cy 3-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-BHQ 1-3'; the probe was labeled with Cy3 at the 5 'end and BHQ1 at the 3' end, and the excitation wavelength of the fluorophore was 552nm and the detection wavelength was 570 nm.

The molecular beacon probes of the invention can be used to detect a wide range of samples including, but not limited to, sputum, pharyngeal swabs, gastric lavage, bronchial lavage, biopsy, aspirates, expectorates, body fluids (spinal cord, pleural effusion, pericardial fluid, etc.), blood, pus, bone marrow, urine, tissue sections, food samples, samples from soil, air and water, and cultures thereof. After appropriate treatment of these samples, the molecular beacon probes of the invention can be used in principle as long as the cell morphology remains intact and the target nucleic acid is not destroyed. Methods of processing these samples are well within the skill of the art, for example:

phlegm: sputum smear method;

pus: smear with sputum;

focal tissue: firstly, grinding by using a tissue grinder and then smearing;

urine: remaining the whole amount of nocturia, standing for 4-5 h, removing supernatant, taking 10mL of sediment part urine, centrifuging at 3000rpm for 30min, and taking a sediment smear;

hydrothorax and ascites specimens: reference urine smear method;

cerebrospinal fluid: collecting cerebrospinal fluid by aseptic operation, placing in refrigerator or room temperature for 24h, and smearing after pellicle is formed. Or centrifuging cerebrospinal fluid at 3000rpm for 30min, discarding supernatant, and collecting precipitate smear.

The molecular beacon probe has high signal intensity and high specificity, and can effectively and quickly detect septicemia.

Detailed Description

The embodiments of the present invention will be described in detail with reference to examples, but the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The examples do not show the specific conditions, and the conventional conditions or the conditions recommended by the manufacturer are followed. The reagents or instruments used are conventional products which are commercially available, not indicated by the manufacturer.

Example 1: design and synthesis of molecular beacon probes and oligonucleotide sequences

Selecting a target sequence capable of specifically detecting septicemia, designing a molecular beacon probe completely complementary to the target sequence on the target sequence: beacon SEP: (5 '-Cy 3-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-BHQ 1-3');

the molecular beacon is a specific sequence of a neck-ring structure consisting of bases, in which the 5 ' -end of the probe is labeled with Cy3, which is a perfectly complementary oligonucleotide (5'-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-3'). By performing thermal denaturation curve experiments on molecular beacons and oligonucleotides, the optimal reaction temperature for fluorescence in situ hybridization is determined to be 52 ℃, and the optimal concentration of the deionized formamide is determined to be 20%.

Example 2: detecting septicemia standard strain by using molecular beacon probe FISH.

The sepsis standard strain was purchased from ATCC and tested for 5 sepsis pathogens and 9 other bacteria in total.

The detection kit comprises:

(1) lysis solution: 4% (w/v) sodium hydroxide;

(2) hybridization solution: 10% (w/v) dextran sulfate, 10mM NaCl, 20% (v/v) formamide, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v) polyvinyl and pyrrolidone, 0.2% polysorbate, 5mM Na2EDTA, 0.1% (v/v) Triton X-100, pH 10.

(3) Stopping liquid: 1% (v/v) dilute methanesulfonic acid;

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

The detection method comprises the following steps:

(1) sucking 10 mul of whole blood sample, dripping the whole blood sample on a glass slide, and naturally drying;

(2) adding 10 mul of lysis solution into the air-dried sample, naturally air-drying, soaking in absolute ethyl alcohol for 5 minutes;

(3) adding 10 mul of hybridization solution to the sample, and placing the sample in a hybridization furnace for hybridization for 10 minutes at 52 ℃;

(4) soaking for 1 minute by using a stop solution, and stopping the reaction;

(5) after addition of the encapsulated tablets, they were examined by fluorescence microscopy, scanned and counted with a 20X objective and observed for bacterial morphology with a 60X or 100X objective. In a dark background, sepsis fluoresces red.

And a result judgment method comprises the following steps:

septicemia pathogenic bacteria (-): 50 different visual fields are observed continuously, and no septicemia pathogenic bacteria are found.

Septicemia pathogenic bacteria (number of reported bacilli): 1-9 strips/50 field.

Septicemia pathogenic bacteria (1 +): 10-99 strips/50 field of view.

Septicemia pathogenic bacteria (2 +): 1-9 per field of view.

Septicemia pathogenic bacteria (3 +): 10-99 strips per field of view.

Pathogenic bacteria (+): >100 strips per field of view

The results are shown in Table 1.

Table 1 detection of septicemia pathogenic bacterial strains using septicemia molecular beacon probe FISH:

and (3) analyzing a detection result: the detection results of 5 kinds of septicemia pathogenic bacteria are all positive, and the detection results of 9 kinds of other bacteria are all negative, which shows that the detection method has good specificity for detecting septicemia, and ensures the reliability and effectiveness of the results. The specificity is embodied based on the following specific probe sequences: beacon SEP: (5'-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-3').

The hybridization reaction time in the experiment only needs 10 minutes, and the whole experiment process can be completed within 1.5 hours, so that the hybridization reaction is very quick. Compared with a plurality of days required by a 'gold standard' blood culture method for detecting septicemia, the method disclosed by the invention has the obvious advantage of rapidness.

It should be understood that the above-mentioned embodiments are only examples for clearly illustrating the present invention, and are not to be construed as limiting the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And it is therefore expressly intended that such changes or modifications are within the scope of the present invention.

The invention refers to the fluorescent label at the 5 'end of the molecular beacon probe, including but not limited to FITC, FAM or Cy3, etc., the fluorescent quenching label at the 3' end, including but not limited to DABCYL, BDH, BHQ1 or TAMRA, etc., the fluorescent group or the fluorescent quenching group can be added according to the prior art; the fluorescent group emission wavelength is 552nm, and the detection wavelength is 570 nm. Other changes within the scope of the present application are common fluorescent markers, and the replacement of the marker has no influence on the experimental results and determination, and can achieve the technical effects obtained by the above embodiments of the present invention.

Sequence listing

<110> Ningbo Meikang Shengde medical laboratory Co., Ltd

<120> molecular beacon probe for detecting septicemia, kit and detection method thereof

<130>2019

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<170>SIPOSequenceListing 1.0

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<211>42

<212>DNA

<213> Artificial sequence ()

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ttccacaaac aggattagat accctggtag tccacgctgg aa 42

Claims (8)

1. A molecular beacon probe for detecting septicemia is characterized in that the base sequence of the molecular beacon probe is as follows: 5'-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-3'; .

2. The molecular beacon probe for detecting septicemia according to claim 1, wherein the molecular beacon probe has a fluorescent label at its 5' end, including but not limited to FITC, FAM or Cy 3; a fluorescence quenching label at the 3' end, including but not limited to DABCYL, BDH, BHQ1 or TAMRA; the fluorescent group emission wavelength is 552nm, and the detection wavelength is 570 nm.

3. The molecular beacon probe for detecting septicemia according to claim 2, wherein the fluorescence label of the 5 'end of the molecular beacon probe is Cy3, the fluorescence quenching of the 3' end is BHQ1, and the base sequence of the corresponding molecular beacon probe is 5 '-Cy 3-TTCCACAAACAGGATTAGATACCCTGGTAGTCCACGCTGGAA-BHQ 1-3'.

4. A kit comprising the molecular beacon probe for detecting septicemia according to any one of claims 1-2, wherein the kit is capable of detecting septicemia.

5. The kit of claim 4, further comprising a lysis solution, a hybridization solution, a stop solution, and a washing solution.

6. The kit according to claim 5,

the lysis solution is 4% (w/v) sodium hydroxide;

the hybridization solution is as follows: 8-12% (w/v) dextran sulfate, 8-12 mM NaCl, 16-24% (v/v) formamide, 0.08-0.12% (w/v) sodium pyrophosphate, 0.16-0.24% (w/v) polyvinylpyrrolidone, 0.16-0.24% (w/v) polyglucoside, 4-6 mM Na₂EDTA，0.08～0.12％(v/v)Triton X-100，40～60mM Tris/HCl(pH7.5)；

The stop solution is 0.08-0.12% (v/v) dilute sulfuric acid;

the washing solution is 4-6 mM Tris, 14-16 mM NaCl, 0.08-0.12% (v/v) Triton X-100, and the pH value is 9.5-10.5.

7. The kit according to claim 6, wherein the concentration of the molecular beacon probe in the kit is 5-20 ng/μ l.

8. A method for rapidly detecting septicemia by using a kit is characterized by comprising the following steps:

(1) dripping 10 mul of sample on a glass slide, and naturally drying;

(2) adding 10 μ l of lysis solution into the air dried sample, air drying, soaking in anhydrous ethanol for 5 min;

(3) adding 8-12 mul of hybridization solution and a molecular beacon probe into a sample, and placing the sample in a hybridization furnace for hybridization for 8-12 minutes at 52 ℃;

(4) soaking for 1 minute by using a stop solution, and stopping the reaction;

(5) and (5) washing with a washing solution.

(6) After addition of the encapsulated tablets, they were examined by fluorescence microscopy, scanned and counted with a 20X objective and observed for bacterial morphology with a 60 or 100X objective.