阅读说明：本技术 一种绿脓杆菌胶体金检测卡及检测方法 (Pseudomonas aeruginosa colloidal gold detection card and detection method ) 是由 甄俊杰 吴靖华 龚俊宇 于 2019-06-16 设计创作，主要内容包括：本发明属于传染病检测领域,涉及一种绿脓杆菌胶体金检测卡及检测方法。本发明所述的胶体金检测卡包括试纸条以及检测卡外壳,所述试纸条包括底板、硝酸纤维素膜、结合垫、吸水纸以及样品垫；所述硝酸纤维素膜粘结在所述底板的中部；所述硝酸纤维素膜的一端粘贴有结合垫,另一端粘贴有吸水纸,所述结合垫上粘贴有样品垫；所述试纸条安装在长条扁平壳状的检测卡外壳中,检测卡外壳上设有加样窗口和检测结果显示窗口,样品垫位于加样窗口,硝酸纤维素膜位于检测结果显示窗口。本发明所述的一种绿脓杆菌胶体金检测卡能够快速检测绿脓杆菌,且具有灵敏度高、特异性强的优点。(The invention belongs to the field of infectious disease detection, and relates to a pseudomonas aeruginosa colloidal gold detection card and a detection method. The colloidal gold detection card comprises a test strip and a detection card shell, wherein the test strip comprises a bottom plate, a nitrocellulose membrane, a combination pad, absorbent paper and a sample pad; the nitrocellulose membrane is bonded in the middle of the bottom plate; one end of the nitrocellulose membrane is pasted with a combination pad, the other end of the nitrocellulose membrane is pasted with absorbent paper, and the combination pad is pasted with a sample pad; the test strip is arranged in a strip flat shell-shaped detection card shell, a sample adding window and a detection result display window are arranged on the detection card shell, the sample pad is positioned in the sample adding window, and the nitrocellulose membrane is positioned in the detection result display window. The colloidal gold detection card for pseudomonas aeruginosa can be used for rapidly detecting pseudomonas aeruginosa and has the advantages of high sensitivity and strong specificity.)

1. The pseudomonas aeruginosa colloidal gold detection card comprises a test strip and a detection card shell, wherein the test strip comprises a bottom plate, a nitrocellulose membrane, a combination pad, absorbent paper and a sample pad; the nitrocellulose membrane is adhered to the middle part of the bottom plate; one end of the nitrocellulose membrane is pasted with a combination pad, the other end of the nitrocellulose membrane is pasted with absorbent paper, and the combination pad is pasted with a sample pad; the test strip is arranged in a strip flat shell-shaped detection card shell, a sample adding window and a detection result display window are arranged on the detection card shell, the sample pad is positioned in the sample adding window, and the nitrocellulose membrane is positioned in the detection result display window.

2. The colloidal gold assay card of claim 1, wherein the nitrocellulose membrane is provided with a test line and a control line, the test line is sprayed with a capture probe, and the control line is sprayed with a biotinylated rabbit polyclonal antibody; the test line is located and is close to combination pad one side, the control line is located and is close to absorbent paper one side.

3. A colloidal gold assay card as claimed in claim 1 wherein the conjugate pad has immobilized thereon colloidal gold-labeled streptavidin, and the conjugate pad has gold nanoparticles of 15 to 20nm in diameter.

4. A colloidal gold assay card as defined in claim 1 wherein said sample pad is prepared by the following method: the glass fibers were soaked in a solution containing 1% BSA, 2% Triton, 2% PEG4000, 20 mM Tris-Ac and 50mM NaAc, air dried, and stored at 4 ℃ until use.

5. A colloidal gold assay card as claimed in claim 1 wherein the capture probe is a single stranded DNA complementary to a reporter single stranded reporter molecule.

6. A method for detecting a colloidal gold assay card according to claim 1, comprising the steps of:

amplifying target DNA or whole cells of pseudomonas aeruginosa by an LAMP method and detecting a Cas12a system, dropwise adding the obtained solution serving as a detection sample onto a sample adding window of the colloidal gold detection card of claim 1 within 5 minutes, and observing the result by naked eyes or reading the result by a portable colloidal gold card reader;

the LAMP method comprises the following steps: the target DNA of Pseudomonas aeruginosa or the whole cell is added to the reaction system (four primers F3, B3, FIB, FIP, dNTP, MgSO)₄(ii) a 1 × isothermal amplification buffer solution), heating at 95 ℃ for 5min, cooling, adding 8U Bst DNA polymerase, preserving the heat at 65 ℃ for 15-30min, heating at 80 ℃ for 10min, and terminating the reaction to obtain an amplification product;

the detection method of the Cas12a system comprises the following steps: LbaCas12a-crRNA activator mixture (12 μ L1 Xsplit buffer (150 mM KCl, 10 mM MgCl)₂After 1% glycerol, 0.5 mM DTT, 20nM HEPES (pH 7.5)), 3 μ L crRNA (300 nM), 1 μ L LbaCas12a (36 nM) and 5 μ L activator (40 nM)) are reacted at 37 ℃ for 30min, 2 μ L of the biotinylated reporter (20 nM) and 2 μ L of the amplified LAMP product are added, and after uniform mixing, the reaction is reacted at 37 ℃ for 15min, and then heated at 98 ℃ for 2min, and the reaction is terminated.

Technical Field

The invention belongs to the field of infectious disease detection, and particularly relates to a pseudomonas aeruginosa colloidal gold detection card and a detection method.

Background

Pseudomonas aeruginosa (p. aeruginosa) or pseudomonas aeruginosa is a less pathogenic but highly drug resistant bacillus. It is widely existed in nature, is a common bacterium of wound infection, can cause suppurative lesion, and is named because pus and exudate are green after infection. Pseudomonas aeruginosa (p. aeruginosa) belongs to the genus Pseudomonas, and is widely distributed in the natural world as well as in normal human skin, intestinal tract and respiratory tract. When the human body has low immune function (such as organ transplantation, leucopenia caused by various reasons, long-term use of adrenocortical hormone, repeated application of a large amount of antibiotics and the like), the human body is easy to be infected by the human body.

Pseudomonas aeruginosa is one of the main pathogenic bacteria in clinical examination. The world health organization (WTO) uses this strain as a pollution hazard indicator for bottled water, and japanese, uk, canada, and other countries have made restrictions on the detection of pseudomonas aeruginosa in bottled mineral water. In recent years, pseudomonas aeruginosa is frequently outbreaked in livestock and poultry breeding and can be infected by fruits and vegetables, and the pseudomonas aeruginosa is identified as a food-borne and water-borne pathogen and is a great threat to human health. Therefore, it is imperative to establish a feasible detection method for pseudomonas aeruginosa.

The CRISPR system is a bacterial immune system that has been found to be present in most bacteria and is used to recognize and destroy phage and other pathogen invasion. CRISPR is a unique DNA region in the bacterial genome that stores viral DNA fragments, allowing bacterial cells to recognize viruses that attempt to re-infect it. The short RNA sequence (referred to as crRNA) generated after transcription of the CRISPR region sequence, upon recognition and binding of the viral nucleic acid, results in cleavage of the viral nucleic acid by a Cas protein (or referred to as Cas enzyme) bound to the crRNA. Various CRISPR/Cas systems have been discovered so far, such as CRISPR/Cas9 and CRISPR/Cas12 a. Unlike Cas9, Cas12a has a corresponding nuclease activity upon activation, in addition to being able to cleave the target nucleic acid, it also has a complimentary cleavage (collatoral cleavage) activity, being able to continue to cleave nearby other non-target single-stranded DNA. Based on these features, Cas12a can be used for detection of target nucleic acids in a sample. However, the sensitivity is difficult to satisfy the detection requirement. The LAMP detection technology is used as a new method based on nucleic acid amplification, provides great help for identifying microbial pathogens since the emergence of 2000, has simple operation method and short reaction time, can complete the reaction only in a constant-temperature water bath kettle, does not need an expensive PCR instrument for temperature circulation compared with the PCR method, and greatly reduces the detection cost.

The LAMP amplification technology, the Cas12a detection technology and the colloidal gold test strip technology are combined together, so that rapid, high-specificity and high-sensitivity detection of pseudomonas aeruginosa is realized.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides the pseudomonas aeruginosa colloidal gold detection card and the detection method, and the pseudomonas aeruginosa colloidal gold detection card and the detection method have the advantages of convenience in operation, rapidness in detection, high specificity, strong sensitivity and the like.

Firstly, the invention provides a pseudomonas aeruginosa colloidal gold detection card, which comprises a test strip and a detection card shell, wherein the test strip comprises a bottom plate, a nitrocellulose membrane, a combination pad, absorbent paper and a sample pad; the nitrocellulose membrane is bonded in the middle of the bottom plate; one end of the nitrocellulose membrane is pasted with a combination pad, the other end of the nitrocellulose membrane is pasted with absorbent paper, and the combination pad is pasted with a sample pad; the test strip is arranged in a strip flat shell-shaped detection card shell, a sample adding window and a detection result display window are arranged on the detection card shell, the sample pad is positioned in the sample adding window, and the nitrocellulose membrane is positioned in the detection result display window.

Preferably, the nitrocellulose membrane is provided with a test line and a control line, the test line is sprayed with a capture probe, and the control line is sprayed with a biotinylated rabbit polyclonal antibody; the test line is located and is close to combination pad one side, the control line is located and is close to absorbent paper one side.

Preferably, the binding pad is fixed with colloidal gold labeled streptavidin, and the diameter of the gold nanoparticle on the binding pad is 15-20 nm.

Preferably, the sample pad is prepared as follows: the glass fibers were soaked in a solution containing 1% BSA, 2% Triton, 2% PEG4000, 20 mM Tris-Ac and 50mM NaAc, air-dried, and stored at 4 ℃ until use.

Preferably, the capture probe is a single-stranded DNA complementary to the reporter single-stranded reporter.

Preferably, the colloidal gold-labeled streptavidin is prepared as follows:

(1) adding 100 mL of 1 mM chloroauric acid solution into a 350mL round-bottom flask, and heating to boil with magnetic stirring; and then adding 3.5 mL of 1% sodium citrate into the solution, continuing to boil for 10min after the solution turns to wine red, stopping heating, continuing to stir for 15min to obtain a colloidal gold AuNP solution, and storing the colloidal gold AuNP solution at 4 ℃ in a dark place.

(2) Adding 100 mu L of suspension buffer (20 mM Na) to 1mL of the colloidal gold solution prepared in the step (1)₃PO₄5% BSA, 0.25% Tween-20, 10% sucrose, and 0.1% NaN₃) After 5min, 0.5 mg/mL SA (streptavidin) was added and the mixture was shaken at 4 ℃ for 3 hours. Blocking was performed by adding 10% BSA (100. mu.L) for 30min, centrifuging at 4 ℃ (12000 rpm, 25 min), and washing with the suspension buffer to remove unbound SA, resulting in AuNP-SA (colloidal gold-labeled streptavidin) probe. Discard the supernatant and resuspend the AuNP-SA probe with 100. mu.L suspension buffer.

Secondly, the detection method of the pseudomonas aeruginosa colloidal gold detection card comprises the following operation steps:

amplifying target DNA or whole cells of pseudomonas aeruginosa by an LAMP method and detecting a Cas12a system, dropwise adding the obtained solution serving as a detection sample onto a sample adding window of the colloidal gold detection card within 5 minutes, and observing the result by naked eyes or reading the result by using a portable colloidal gold card reader;

preferably, the LAMP method is as follows: the target DNA of Pseudomonas aeruginosa or the whole cell is added to the reaction system (four primers F3, B3, FIB, FIP, dNTP, MgSO)₄(ii) a 1 × isothermal amplification buffer solution), heating at 95 ℃ for 5min, cooling, adding 8U Bst DNA polymerase, preserving the heat at 65 ℃ for 15-30min, heating at 80 ℃ for 10min, and terminating the reaction to obtain an amplification product;

preferably, the Cas12a system detection method is as follows: LbaCas12a-crRNA activator mixture (12 μ L1 Xsplit buffer (150 mM KCl, 10 mM MgCl)₂，1 % glycerol，0.5 mM DTT，20 nMHEPES (pH 7.5)), 3 μ L crRNA (300 nM), 1 μ L LbaCas12a (36 nM) and 5 μ L activator (40 nM)) were reacted at 37 ℃ for 30min, 2 μ L biotinylated reporter (20 nM) and 2 μ L LAMP amplification product were added, after uniform mixing, reacted at 37 ℃ for 15min, and then heated at 98 ℃ for 2min, and the reaction was terminated.

Compared with the prior art, the invention has the advantages and beneficial effects that:

(1) the Pseudomonas aeruginosa colloidal gold detection card provided by the invention is convenient to operate, does not need special experimental conditions, can be used for clinical examination by observing results with naked eyes within 5 minutes or reading the results with a portable colloidal gold card reader, and has a good application prospect in the field of medical treatment.

(2) The colloidal gold detection card for pseudomonas aeruginosa has strong specificity in detecting pseudomonas aeruginosa, and other germs do not interfere with detection signals. Only when there is Pseudomonas aeruginosa cell or target DNA in the sample, the reporter single-stranded reporter is cleaved and cannot be captured by the capture probe coated on the test line.

(3) The colloidal gold detection card for pseudomonas aeruginosa has high sensitivity in determination of pseudomonas aeruginosa. After the pseudomonas aeruginosa is subjected to LAMP amplification, the detection is carried out, so that the detection sensitivity can be obviously improved.

Drawings

Fig. 1 is a schematic side structure view of a pseudomonas aeruginosa colloidal gold test strip of the present invention.

FIG. 2 is a schematic diagram of the detection principle of the Pseudomonas aeruginosa colloidal gold detection card according to the present invention.

FIG. 3 is a diagram showing the result of detecting the sensitivity of the Pseudomonas aeruginosa colloidal gold card according to the present invention.

FIG. 4 is a diagram showing the specific detection result of the Pseudomonas aeruginosa colloidal gold detection card according to the present invention.

FIG. 5 is a schematic diagram of the detection result of the Pseudomonas aeruginosa colloidal gold test card on the actual sample.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.