阅读说明：本技术 一种利用培养法检测解脲脲原体和微小脲原体的试剂盒及其制备方法 (Kit for detecting ureaplasma urealyticum and ureaplasma parvum by using culture method and preparation method thereof ) 是由 周丽霞 王则宇 王利英 李晓霞 张利红 刘亚峰 秦军领 崔晓 于 2020-06-05 设计创作，主要内容包括：本发明涉及临床诊断技术领域,公开了一种利用培养法检测解脲脲原体和微小脲原体的试剂盒及其制备方法。本发明所述试剂盒包括载有微小脲原体鉴定孔和解脲脲原体鉴定孔的检测板；其中,所述解脲脲原体鉴定孔中含有脲酶抑制剂和抗菌药物。本发明提供了一种新型用培养法鉴别区分解脲脲原体(UU)和微小脲原体(UP)试剂盒,将脲酶抑制剂和抗菌药物联合使用,对解脲脲原体(UU)鉴定孔的特异性有明显的提高,从而将脲原体属中的解脲脲原体(UU)和微小脲原体(UP)彻底分开,同时某些抗菌药物组合可以提高其检测灵敏度。本发明试剂盒操作简单方便,适于临床的大面积普及应用。(The invention relates to the technical field of clinical diagnosis, and discloses a kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method and a preparation method thereof. The kit comprises a detection plate carrying a small ureaplasma urealyticum identification hole and a ureaplasma urealyticum identification hole; wherein, the ureaplasma urealyticum identification hole contains urease inhibitor and antibacterial drug. The invention provides a novel kit for identifying and distinguishing Ureaplasma Urealyticum (UU) and ureaplasma micans (UP) by using a culture method, wherein a urease inhibitor and an antibacterial agent are combined for use, the specificity of a Ureaplasma Urealyticum (UU) identification hole is obviously improved, so that the Ureaplasma Urealyticum (UU) and the ureaplasma micum micans (UP) in ureaplasma are thoroughly separated, and meanwhile, the detection sensitivity of some antibacterial agent combinations can be improved. The kit is simple and convenient to operate and is suitable for large-area popularization and application in clinic.)

1. A kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method is characterized by comprising a detection plate carrying a ureaplasma urealyticum identification hole and a ureaplasma urealyticum identification hole; wherein, the ureaplasma urealyticum identification hole contains urease inhibitor and antibacterial drug.

2. The kit of claim 1, wherein the urease inhibitor is selected from the group consisting of hydroxamates and phosphoramidates.

3. The kit according to claim 1 or 2, wherein the urease inhibitor is selected from one or more of acetohydroxamic acid, n-butyl thiophosphoric triamide and butyl phosphoric triamide.

4. The kit according to claim 1, wherein the antibacterial drug is one or more selected from the group consisting of levofloxacin, moxifloxacin, gentamicin, pristinamycin and amikacin.

5. The kit of claim 1 or 4, wherein the antibacterial drug is a combination of pristinamycin + gentamicin, moxifloxacin + gentamicin, or levofloxacin + gentamicin.

6. The kit of claim 1, wherein the urease inhibitor is present in an amount ranging from 2mg/L to 200 mg/L.

7. The kit of claim 1, wherein the antibacterial agent is present in an amount of 1mg/L to 50 mg/L.

8. The kit according to claim 1, wherein the ureaplasma parvum identification well contains 165mg/L to 550mg/L of manganese ions.

9. The kit of claim 8, wherein the manganese ion is provided by a manganese sulfate, chloride, nitrate or acetate solution.

10. The method for preparing the kit according to claim 1, wherein the kit is obtained by adding the urease inhibitor and the antibacterial agent into the ureaplasma urealyticum identification well at a concentration of 20 μ L/well as adding the manganese ion solution into the ureaplasma urealyticum identification well at a concentration of 20 μ L/well, and then drying both wells to attach the urease inhibitor and the antibacterial agent to the inner wall of the ureaplasma urealyticum identification well and to attach the manganese ions to the inner wall of the ureaplasma urealyticum identification well.

Technical Field

The invention relates to the field of clinical diagnosis, in particular to a kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method and a preparation method thereof.

Background

In 1999, Kong et al, based on phylogenetic analysis, divided two biotypes of Ureaplasma into two new species, with organism group i as an independent species, named Ureaplasma Parvum (UP); the organism group II still retains the original name Ureaplasma Urealyticum (UU). Until 2007, the international society for bacteriology taxonomy formally identified organism group i in ureaplasma as a new species, namely Ureaplasma Parvum (UP). The research on the relevance of the Ureaplasma Urealyticum (UU) to various diseases is carried out on the basis of the urea Ureaplasma Parvum (UP) and the urea Ureaplasma Urealyticum (UU) for a long time, so that the urea Ureaplasma Urealyticum (UU) clinically presents an ultrahigh positive rate but has low relevance to the diseases, and the clinical detection value and the clinical detection meaning of the urea Ureaplasma Urealyticum (UU) are questioned.

The current consensus is that Ureaplasma Urealyticum (UU) is highly related to Nongonorrhealurethritis (NGU), and Ureaplasma Parvum (UP) is more common and asymptomatic. Because the relevance of the Ureaplasma Parvum (UP) and the nongonococcal urethritis NGU is low, and the carrying rate is far higher than that of the Ureaplasma Urealyticum (UU), the accurate detection and identification of the Ureaplasma Urealyticum (UU) are particularly necessary.

The types of ureaplasma urealyticum detection reagents in the market are many, including a culture method, an immunological method and a molecular biological method, but most of the Ureaplasma Urealyticum (UU) and the ureaplasma micrum (UP) are detected, and the ureaplasma urealyticum and the ureaplasma micum cannot be separately detected. At present, only molecular biology methods, common fluorescence PCR combined gel electrophoresis and whole gene sequencing, which can really distinguish and detect Ureaplasma Urealyticum (UU) and Ureaplasma Parvum (UP) independently, are available, but the reagents are often too complex to operate and very expensive, are not suitable for clinical popularization and application, and cannot synchronously perform anti-drug determination.

Disclosure of Invention

In view of the above, the present invention aims to provide a kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method and a preparation method thereof, so that the kit can improve the sensitivity and specificity of detecting ureaplasma urealyticum.

In order to achieve the above purpose, the invention provides the following technical scheme:

a kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method comprises a detection plate carrying ureaplasma urealyticum identification holes and ureaplasma urealyticum identification holes; wherein, the ureaplasma urealyticum identification hole contains urease inhibitor and antibacterial drug.

The invention combines urease inhibitor and antibacterial agent, which improves the specificity of Ureaplasma Urealyticum (UU) identification hole and meets the performance requirement. Thereby completely separating Ureaplasma Urealyticum (UU) and Ureaplasma Parvum (UP) in the genus ureaplasma. The principle of using urease inhibitors in combination with certain antibacterial agents is as follows: (1) the urease inhibitor is selected according to the selection that when urea is decomposed by the ureaplasma micranthum (UP), metal chaperone protein is needed to catalyze and mature the urease, a urease metal chaperone protein blocking agent (namely, the urease inhibitor) is selected, and the maturation process of the urease is destroyed by functional disturbance, so that the urease of the ureaplasma micranthum (UP) loses the urea decomposition capability, and the ureaplasma micranthum (UP) and the Ureaplasma Urealyticum (UU) are distinguished; (2) the antimicrobial agent is selected based on the fact that the Minimum Inhibitory Concentration (MIC) value of Ureaplasma Urealyticum (UU) is higher than that of Ureaplasma Parvum (UP). Meanwhile, the invention also discovers that the detection sensitivity of ureaplasma urealyticum can be improved by selecting a specific antibacterial drug combination on the basis of improving the specificity.

Preferably, the urease inhibitor is selected from the group consisting of hydroxamic acids and phosphoramidates. Further, the urease inhibitor is preferably one or more than two of acetohydroxamic acid, n-butyl thiophosphoric triamide and butyl phosphoric triamide.

Preferably, the antibacterial drug is one or more than two of levofloxacin, moxifloxacin, gentamicin, pristinamycin and amikacin. More preferably, the antibacterial drug is a combination of pristinamycin + moxifloxacin, moxifloxacin + gentamicin or a combination of levofloxacin + gentamicin, most preferably a combination of pristinamycin + moxifloxacin.

Preferably, the content of the urease inhibitor is 2mg/L-200 mg/L. In a specific embodiment of the invention, the urease inhibitor is 5.7mg/L of n-butyl thiophosphoric triamide or 47-53mg/L of acetohydroxamic acid.

Preferably, the content of the antibacterial drug is 1mg/L-50 mg/L. In a specific embodiment of the invention, the antibacterial drug is moxifloxacin not higher than 2mg/L, pristinamycin not higher than 20mg/L, levofloxacin not higher than 2mg/L, gentamicin not higher than 42mg/L or a combination thereof.

Preferably, the ureaplasma parvum identifying well contains 165mg/L-550mg/L (the content of manganese in manganese salt is shown here because manganese salt is different, such as manganese chloride, manganese chloride tetrahydrate, manganese sulfate, manganese acetate, and the like, and the molecular weight is 20 muL/well), at which the manganese ion can inhibit UU without reaching the amount for inhibiting UP. The manganese ion can be any solution capable of providing free manganese ions, and in the embodiment of the invention, the manganese ion is provided by manganese sulfate, manganese chloride, manganese nitrate or manganese acetate solution, and the concentration of the manganese ion can be specifically selected to be 191mg/L, 267mg/L, 178mg/L and 394 mg/L.

Simultaneously, the invention also provides a preparation method of the kit, and the kit is obtained by adding the urease inhibitor and the antibacterial drug into the ureaplasma urealyticum identification hole at the concentration of 20 muL/hole according to the requirement, adding the manganese ion solution into the ureaplasma urealyticum identification hole at the concentration of 20 muL/hole according to the requirement, drying the two holes, attaching the urease inhibitor and the antibacterial drug on the inner wall of the ureaplasma urealyticum identification hole, and attaching the manganese ions on the inner wall of the ureaplasma urealyticum identification hole.

According to the technical scheme, the invention provides a novel kit for identifying and distinguishing Ureaplasma Urealyticum (UU) and ureaplasma micans (UP) by using a culture method, a urease inhibitor and an antibacterial agent are combined for use, the specificity of an identification hole of the Ureaplasma Urealyticum (UU) is obviously improved, so that the Ureaplasma Urealyticum (UU) and the ureaplasma micum micans (UP) in ureaplasma are thoroughly separated, and meanwhile, the detection sensitivity of some antibacterial agent combinations can be improved. The kit is simple and convenient to operate and is suitable for large-area popularization and application in clinic.

Detailed Description

The embodiment of the invention discloses a kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method and a preparation method thereof, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the kit and method of making the same according to the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and practiced with modification, or with appropriate modification, and combination of the kit and method of making the same without departing from the spirit, scope, and spirit of the invention.

When the kit is used, the strain or the suspicious sample is added into a ureaplasma urealyticum culture medium, is uniformly mixed and then is respectively added into a Ureaplasma Urealyticum (UU) identification hole and a ureaplasma micum (UP) identification hole, is cultured for 24-48 hours at 37 ℃, and is used for judging whether the Ureaplasma Urealyticum (UU) and the ureaplasma micum (UP) exist in the sample according to whether the culture medium in the Ureaplasma Urealyticum (UU) identification hole and the ureaplasma micum (UP) identification hole is discolored or not.

The interpretation method comprises the following steps: (1) the Ureaplasma Urealyticum (UU) identification hole and the ureaplasma micracum (UP) identification hole are discolored, so that the ureaplasma urealyticum and the ureaplasma micracum exist in the sample at the same time;

(2) the Ureaplasma Urealyticum (UU) identification hole is discolored, and the ureaplasma micans (UP) identification hole is not discolored, so that the sample only contains ureaplasma urealyticum;

(3) the Ureaplasma Urealyticum (UU) identification hole does not change color, and the ureaplasma micans (UP) identification hole changes color, so that the sample only contains the ureaplasma micans;

(4) and the Ureaplasma Urealyticum (UU) identification hole and the ureaplasma micracum (UP) identification hole are not discolored, so that the sample has no ureaplasma urealyticum or ureaplasma micracum.

The MIC values of UU and UP of the invention are derived from literature and clinical strain tests of ureaplasma urealyticum and ureaplasma parvum of nearly hundred strains, and the MIC values of selected antibacterial agents to UU and UP are shown in the following table 1:

TABLE 1

Antibacterial agent	UU (MIC value)	UP (MIC value)
			Levofloxacin	2mg/L	1mg/L
Moxifloxacin hydrate	2mg/L	0.5mg/L
			Gentamicin	42mg/L	10mg/L
Protomycin	20mg/L	2mg/L
			Amikacin	5mg/L	10mg/L

The kit for detecting ureaplasma urealyticum and ureaplasma parvum by using a culture method and the preparation method thereof provided by the invention are further described below.