CN104762235A - Helicobacter pylori transport culture medium as well as preparation method and application thereof - Google Patents
Helicobacter pylori transport culture medium as well as preparation method and application thereof Download PDFInfo
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- 239000006163 transport media Substances 0.000 claims abstract description 32
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Abstract
本发明涉及微生物学领域，具体涉及一种幽门螺杆菌运送培养基及其制备与应用。所述运送培养基包括琼脂和还原剂等，能有效地保证幽门螺杆菌的一种微需氧环境，因而能尽可能长时间保存临床样本的活性状态，在非培养状态下存活的时间可达48h以上，有利于标本远距离的转运，利于医院和检验人员的灵活安排标本的检验时间。本发明的幽门螺杆菌运送培养基由于能长时间保存样本的活性状态，因而对后续的培养有很好的促进作用，能够避免采集到的幽门螺杆菌变成非可培养态的状态，大大提高阳性检出率。与以往不采用的转运培养基相比，采用本发明的转运培养基，阳性检出率提高40%以上。The invention relates to the field of microbiology, in particular to a transport culture medium for Helicobacter pylori and its preparation and application. The transport medium includes agar and reducing agent, etc., which can effectively ensure a microaerophilic environment for Helicobacter pylori, so that the active state of the clinical sample can be preserved for as long as possible, and the survival time in the non-culture state can reach More than 48 hours is conducive to the long-distance transfer of specimens, and is conducive to the flexible arrangement of specimen inspection time by hospitals and inspectors. Because the Helicobacter pylori transport medium of the present invention can preserve the active state of the sample for a long time, it has a good promotion effect on the subsequent cultivation, and can avoid the collected Helicobacter pylori from becoming a non-culturable state, greatly improving positive detection rate. Compared with the transport medium not used in the past, the positive detection rate is increased by more than 40% by adopting the transport medium of the present invention.
Description
技术领域technical field
本发明涉及微生物学领域，具体涉及一种幽门螺杆菌运送培养基及其制备与应用。The invention relates to the field of microbiology, in particular to a transport culture medium for Helicobacter pylori and its preparation and application.
背景技术Background technique
幽门螺旋杆菌(Helicobacter pylori,Hp)是一种革兰阴性微需氧杆菌。2005年的诺贝尔生理学和医学奖获得者Warren及Marshall在1983从慢性胃炎病人的胃镜活检标本中分离出Hp以来，受到国内外医学界众多学者的高度关注。Hp感染呈全球性分布，Hp和一些上消化道疾病发生有紧密联系，是慢性胃炎、胃十二指肠溃疡的重要致病因素，与胃癌、胃黏膜相关淋巴组织淋巴瘤发生密切相关，世界卫生组织已将其列为一类致病因子。Helicobacter pylori (Hp) is a Gram-negative microaerophilic bacterium. Since Warren and Marshall, winners of the Nobel Prize in Physiology and Medicine in 2005, isolated Hp from gastroscopic biopsy specimens of patients with chronic gastritis in 1983, they have received great attention from many scholars in the medical field at home and abroad. Hp infection is distributed globally, Hp is closely related to some upper gastrointestinal diseases, is an important pathogenic factor of chronic gastritis and gastroduodenal ulcer, and is closely related to gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. The health organization has listed it as a class of pathogenic factors.
现阶段的临床检测，主要有侵入性和非侵入性两大类，前者有细菌培养、快速尿酶试验等，后者有尿呼吸试验、抗体检测等。细菌培养为诊断幽门螺杆菌的金标准。幽门螺杆菌稳定生长需要依赖微需氧环境，其次在培养时需保持环境相对湿度达到90％以上，以及最适生长的温度为37℃，pH值为中性。幽门螺杆菌在自然环境中不能生长繁殖，临床从采集样本到真正开始培养检测还需要一段时间，而这段时间对于幽门螺杆菌的后续培养繁殖是至关重要的，临床上往往由于样本保存不当，即使采用最好的培养基，也会导致阳性检出率大大降低。目前国内对标本的运输保存条件研究得还比较少，或者说是关注度还不高。本发明解决的正是样本从采集后到开始培养的这段时间如何有效地保证幽门螺杆菌存活率的问题。Clinical tests at this stage mainly fall into two categories: invasive and non-invasive. The former includes bacterial culture, rapid urine enzyme test, etc., while the latter includes urine breath test, antibody detection, etc. Bacterial culture is the gold standard for the diagnosis of Helicobacter pylori. The stable growth of Helicobacter pylori needs to rely on a microaerobic environment. Secondly, the relative humidity of the environment must be kept above 90% during cultivation, and the optimum growth temperature is 37°C, and the pH value is neutral. Helicobacter pylori cannot grow and reproduce in the natural environment. It will take some time from the collection of samples to the actual start of culture and detection in clinical practice, and this period of time is crucial for the subsequent culture and reproduction of Helicobacter pylori. , even with the best culture medium, the positive detection rate will be greatly reduced. At present, there is still relatively little research on the transportation and preservation conditions of specimens in China, or the degree of attention is not high. The present invention solves the problem of how to effectively ensure the survival rate of Helicobacter pylori during the period from sample collection to initiation of culture.
发明内容Contents of the invention
针对现有技术中所存在的缺陷，本发明的目的在于，提供一种幽门螺杆菌运送培养基，以长时间地保存所采集到的种幽门螺杆菌样本，提高幽门螺杆菌的阳性检出率。For the defects existing in the prior art, the object of the present invention is to provide a Helicobacter pylori transport culture medium to preserve the collected Helicobacter pylori samples for a long time and improve the positive detection rate of Helicobacter pylori. .
本发明的另一目的在于，提供所述幽门螺杆菌运送培养基的制备方法及其应用。Another object of the present invention is to provide the preparation method and application of the Helicobacter pylori transport medium.
本发明的再一目的在于，提供一种提高幽门螺杆菌的阳性检出率的方法。Another object of the present invention is to provide a method for increasing the positive detection rate of Helicobacter pylori.
本发明是通过以下技术方案实现的：The present invention is achieved through the following technical solutions:
本发明通过在琼脂中添加还原剂，研发出一种能够提供适合幽门螺杆菌稳定生长所需气体环境和营养成分的运送培养基。The present invention develops a transport medium capable of providing the gas environment and nutrient components suitable for the stable growth of Helicobacter pylori by adding a reducing agent to the agar.
本发明的第一方面提供了一种幽门螺杆菌运送培养基，每1000ml所述幽门螺杆菌运送培养基中含有：琼脂3.0-8.0g；抗坏血酸5.0-15.0mg；葡萄糖5.0-10.0g；甘氨酸10.0-50.0mg；二硫苏糖醇5.0-15.0mg；硫酸亚铁铵3.0-8.0mg；硼氢化钠3.0-8.0mg。The first aspect of the present invention provides a transport medium for Helicobacter pylori, which contains: 3.0-8.0 g of agar; 5.0-15.0 mg of ascorbic acid; 5.0-10.0 g of glucose; 10.0 g of glycine -50.0mg; dithiothreitol 5.0-15.0mg; ferrous ammonium sulfate 3.0-8.0mg; sodium borohydride 3.0-8.0mg.
优选地，每1000ml所述幽门螺杆菌运送培养基中含有：琼脂5.0g。Preferably, every 1000ml of the Helicobacter pylori transport medium contains: 5.0g of agar.
优选地，每1000ml所述幽门螺杆菌运送培养基中含有：琼脂5.0g，葡萄糖7.0g，二硫苏糖醇10.0mg。Preferably, every 1000 ml of the Helicobacter pylori transport medium contains: 5.0 g of agar, 7.0 g of glucose, and 10.0 mg of dithiothreitol.
更优选地，每1000ml所述幽门螺杆菌运送培养基中含有：琼脂5.0g；抗坏血酸10mg；葡萄糖7.0g；甘氨酸30.0mg；二硫苏糖醇10.0mg；硫酸亚铁铵5.0mg；硼氢化钠5.0mg。More preferably, every 1000ml of the Helicobacter pylori transport medium contains: agar 5.0g; ascorbic acid 10mg; glucose 7.0g; glycine 30.0mg; dithiothreitol 10.0mg; ferrous ammonium sulfate 5.0mg; 5.0 mg.
本发明第二方面提供了一种制备所述幽门螺杆菌运送培养基的方法，包括步骤：按重量份称取各组分，加入至去离子水中，溶解，高压灭菌，分装即可。The second aspect of the present invention provides a method for preparing the transport culture medium for Helicobacter pylori, comprising the steps of: weighing each component by weight, adding to deionized water, dissolving, autoclaving, and subpackaging.
本发明第三方面提供了所述幽门螺杆菌运送培养基在运送幽门螺杆菌中的用途。The third aspect of the present invention provides the use of the Helicobacter pylori transport medium in transporting Helicobacter pylori.
优选地，所述用途为将所述幽门螺杆菌运送培养基用于运送幽门螺杆菌样本以提高幽门螺杆菌阳性检出率。Preferably, the use is to use the Helicobacter pylori transport medium to transport Helicobacter pylori samples to increase the positive detection rate of Helicobacter pylori.
本发明的第四方面提供了一种运送幽门螺杆菌样本的方法，包括步骤：将采集的幽门螺杆菌样本放入所述幽门螺杆菌运送培养基中运送。The fourth aspect of the present invention provides a method for transporting a Helicobacter pylori sample, comprising the step of: putting the collected Helicobacter pylori sample into the Helicobacter pylori transport medium for transport.
本发明的第五方面还提供了一种提高幽门螺杆菌阳性检出率的方法，包括步骤：将采集的幽门螺杆菌样本放入前述幽门螺杆菌运送培养基运送，再进行后续的培养检测。The fifth aspect of the present invention also provides a method for increasing the positive detection rate of Helicobacter pylori, comprising the steps of: putting the collected Helicobacter pylori sample into the aforementioned Helicobacter pylori transport medium for transport, and then performing subsequent culture and detection.
本发明的有益效果在于：The beneficial effects of the present invention are:
(1)本发明的幽门螺杆菌转运培养基配制简单，成本低廉。(1) The Helicobacter pylori transport medium of the present invention is simple to prepare and low in cost.
(2)本发明的幽门螺杆菌运送培养基为半固体培养基，运输方便，易携带。克服了液体状的培养基在运输过程中容易与瓶口接触，导致开盖关盖染菌几率大大提高的缺陷。而本发明的半固体的培养基由于其流动性差，就大大降低这种染菌情况的发生。(2) The transport culture medium for Helicobacter pylori of the present invention is a semi-solid culture medium, which is convenient for transportation and easy to carry. It overcomes the defect that the liquid culture medium is easy to contact with the mouth of the bottle during transportation, which greatly increases the probability of bacterial infection when the cap is opened and closed. However, the semi-solid culture medium of the present invention greatly reduces the occurrence of this bacterial contamination due to its poor fluidity.
(3)本发明所添加的还原剂，能有效地保证幽门螺杆菌的一种微需氧环境，因而能尽可能长时间保存临床样本的活性状态，在非培养状态下存活的时间可达48h以上，有利于标本远距离的转运，利于医院和检验人员的灵活安排标本的检验时间。(3) The reducing agent added by the present invention can effectively guarantee a microaerophilic environment of Helicobacter pylori, thus the active state of the clinical sample can be preserved for as long as possible, and the survival time in the non-cultured state can reach 48h The above is conducive to the long-distance transfer of specimens, and is conducive to the flexible arrangement of specimen inspection time by hospitals and inspectors.
(4)本发明的幽门螺杆菌运送培养基由于能长时间保存样本的活性状态，因而对后续的培养有很好的促进作用，能够避免采集到的幽门螺杆菌变成非可培养态的状态，大大提高阳性检出率。与以往不采用的转运培养基相比，采用本发明的转运培养基，阳性检出率提高40％以上。(4) Since the Helicobacter pylori transport medium of the present invention can preserve the active state of the sample for a long time, it has a good promotion effect on subsequent cultivation, and can avoid the collected Helicobacter pylori from becoming a non-culturable state , greatly increasing the positive detection rate. Compared with the transport medium not used in the past, the positive detection rate is increased by more than 40% by adopting the transport medium of the present invention.
附图说明Description of drawings
图1：为本发明半固体状的转运培养基示意图。Figure 1: Schematic diagram of the semi-solid transport medium of the present invention.
图2：为本发明实施例5采样具体流程示意图。Fig. 2: It is a schematic diagram of the specific sampling process of Example 5 of the present invention.
具体实施方式Detailed ways
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention.
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, devices and materials of the prior art similar or equivalent to the practice of the present invention.
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the seriesMETHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODSIN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology. These technologies have been fully explained in the existing literature. For details, please refer to MOLECULAR CLONING such as Sambrook: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York，1987and periodic updates；the seriesMETHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODSIN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds .), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
实施例1Example 1
本发明所述的运送培养基配制方法为，按下表组分称量：Transport culture medium preparation method of the present invention is, weighs according to the following table components:
加入1L去离子水中，溶解后121℃，高压灭菌15min，然后分装于每个试管中，检验试管的密封性。Add 1L of deionized water, after dissolving, sterilize at 121°C for 15 minutes under high pressure, then divide into each test tube, and check the tightness of the test tube.
实施例2Example 2
本发明所述的运送培养基配制方法为，按下表组分称量：Transport culture medium preparation method of the present invention is, weighs according to the following table components:
加入1L去离子水中，溶解后121℃，高压灭菌15min，然后分装于每个试管中，检验试管的密封性。Add 1L of deionized water, after dissolving, sterilize at 121°C for 15 minutes under high pressure, then divide into each test tube, and check the tightness of the test tube.
实施例3Example 3
本发明所述的运送培养基配制方法为，按下表组分称量：Transport culture medium preparation method of the present invention is, weighs according to the following table components:
加入1L去离子水中，溶解后121℃，高压灭菌15min，然后分装于每个试管中，检验试管的密封性。Add 1L of deionized water, after dissolving, sterilize at 121°C for 15 minutes under high pressure, then divide into each test tube, and check the tightness of the test tube.
实施例4Example 4
转运管密封性能验证：将配置好的转运培养基2～8℃放置两个月，观察其颜色的变化，如仍为蓝色，则能使用，密封性能验证结果如下表1所示：Verification of sealing performance of transfer tube: place the prepared transfer medium at 2-8°C for two months, and observe its color change. If it is still blue, it can be used. The results of sealing performance verification are shown in Table 1 below:
表1 容器密封性试验表Table 1 Container tightness test table
注：变色硅胶的初始颜色为蓝色，当有外部水汽进入容器时，会因吸水程度不同而渐变为不同程度的粉红色Note: The initial color of the color-changing silica gel is blue. When external water vapor enters the container, it will gradually change to different degrees of pink due to the degree of water absorption.
本发明采用性能测试合格的转运管分装实施例1～3所制备的运送培养基。The present invention adopts the transfer tubes that have passed the performance test to subpackage the transport culture medium prepared in Examples 1-3.
实施例5Example 5
使用本发明实施例1～3制备的运送培养基保存临床新采集的幽门螺杆菌样本。The transport medium prepared in Examples 1-3 of the present invention was used to preserve the clinically newly collected Helicobacter pylori samples.
采样流程：HP转运管(2～8℃保存)，样本采集后用采样棒尖端挑取样本组织，确认组织附着后将附有采样组织一端插入试管底部1/3处(如图1)，然后运送至检验实验室进行检验，具体操作流程如图2所示。Sampling process: HP transfer tube (stored at 2-8°C), after sample collection, use the tip of the sampling stick to pick the sample tissue, after confirming the tissue attachment, insert the end with the sample tissue into the bottom 1/3 of the test tube (as shown in Figure 1), and then Transport to the inspection laboratory for inspection, the specific operation process is shown in Figure 2.
进行检测后，统计结果，如下表2所示：After testing, the statistical results are shown in Table 2 below:
表2 采用实施例1～3转运培养基与否阴阳性统计结果Table 2 Statistical results of negative and positive results of using the transport medium in Examples 1 to 3
由上述结果可知，本发明所添加的还原剂，能有效地保证幽门螺杆菌的一种微需氧环境，因而能尽可能长时间保存临床样本的活性状态，在非培养状态下存活的时间可达48h以上，有利于标本远距离的转运，利于医院和检验人员的灵活安排标本的检验时间。From the above results, it can be seen that the reducing agent added by the present invention can effectively ensure a microaerophilic environment for Helicobacter pylori, thus the active state of the clinical sample can be preserved for as long as possible, and the time of survival in the non-cultured state can be shortened. It is more than 48 hours, which is conducive to the long-distance transfer of specimens, and is conducive to the flexible arrangement of specimen inspection time by hospitals and inspectors.
本发明的幽门螺杆菌运送培养基由于能长时间保存样本的活性状态，因而对后续的培养有很好的促进作用，能够避免采集到的幽门螺杆菌变成非可培养态的状态，大大提高阳性检出率。与以往不采用的转运培养基相比，采用本发明的转运培养基，阳性检出率提高40％以上。Because the Helicobacter pylori transport medium of the present invention can preserve the active state of the sample for a long time, it has a good promotion effect on the subsequent cultivation, and can avoid the collected Helicobacter pylori from becoming a non-culturable state, greatly improving positive detection rate. Compared with the transport medium not used in the past, the positive detection rate is increased by more than 40% by adopting the transport medium of the present invention.
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form and in essence. Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those who are familiar with this profession, without departing from the spirit and scope of the present invention, when they can use the technical content disclosed above to make some changes, modifications and equivalent changes of evolution, are all included in the present invention. Equivalent embodiments; at the same time, all changes, modifications and evolutions of any equivalent changes made to the above-mentioned embodiments according to the substantive technology of the present invention still belong to the scope of the technical solution of the present invention.
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