WO2022178982A1 - 幽门螺杆菌敏感、快速培养菌落形成及其效能评价方法 - Google Patents

幽门螺杆菌敏感、快速培养菌落形成及其效能评价方法 Download PDF

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WO2022178982A1
WO2022178982A1 PCT/CN2021/093000 CN2021093000W WO2022178982A1 WO 2022178982 A1 WO2022178982 A1 WO 2022178982A1 CN 2021093000 W CN2021093000 W CN 2021093000W WO 2022178982 A1 WO2022178982 A1 WO 2022178982A1
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helicobacter pylori
plate
culture
colony
colonies
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谷海瀛
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谷海瀛
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Priority to US18/278,425 priority patent/US20240229099A9/en
Publication of WO2022178982A1 publication Critical patent/WO2022178982A1/zh

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Definitions

  • the present invention relates to microbial culture technology, in particular, to a method for evaluating the sensitivity and rapid culture of Helicobacter pylori colony formation and its efficacy.
  • Helicobacter pylori is a microaerophilic bacterium that can continuously colonize human gastrointestinal mucosa and cause various gastritis, duodenitis and peptic ulcer.
  • Helicobacter pylori infection rate can reach more than 50%; and it is also related to parenteral infections, such as pneumonia, bacteremia, and serum glycosylated hemoglobin levels. Therefore, it is necessary to diagnose Helicobacter pylori infection.
  • There are various techniques for diagnosing Helicobacter pylori infection divided into screening tests and confirmatory tests.
  • Screening tests include urease test, histomorphological examination, UBT test, serum antibodies, fecal antigens and molecular biology techniques, etc.; the only diagnostic tests are the isolation and culture of Helicobacter pylori and immunohistochemical techniques, and the isolation and culture of Helicobacter pylori are all diagnostic tests The most technically specific method. Routine pathomorphological diagnosis does not rule out colonization or infection with Campylobacter and other Helicobacter species similar to H. pylori. UBT and urease tests can produce false-positive results due to the presence of other urease-positive bacteria in the stomach, serum antibodies may reflect past infection, poor fecal antigen sensitivity, high molecular biology techniques, and none of these techniques provide reliable drug susceptibility test results.
  • Helicobacter pylori isolation and culture is the "gold standard" for these diagnostic methods and is an essential procedure for phenotypic susceptibility testing and genotyping. In order to prevent and treat diseases caused by Helicobacter pylori infection, it is of great significance to quickly isolate and culture Helicobacter pylori.
  • Culture is divided into primary isolation and subculture. Colony formation includes colony formation from primary isolation and subculture from human specimens. The primary isolation is to inoculate a tissue specimen containing viable H. pylori cells in a suitable isolation medium (primary culture) and allow the cells to grow as many colonies as possible. Subculture is to inoculate a culture of Helicobacter pylori in a subculture medium and allow it to grow well.
  • the isolation medium and subculture medium should be different. Tissue specimens usually contain other bacteria than Helicobacter pylori. Antibiotics that inhibit the growth of bacteria need to be added to the isolation medium without affecting the growth of Helicobacter pylori.
  • the passage medium does not need to add antibiotics, and other components can also be different from the separation medium, which is mainly used for the biological characteristics analysis of Helicobacter pylori, phenotypic drug sensitivity test and other tests.
  • Subculture includes growth of colonies on plate media and revived colonies from cryopreserved cultures. The resurrection rate of subculture is affected by factors such as medium composition and stability. There are two important factors affecting the initial isolation results of Helicobacter pylori: recovery rates and isolation time.
  • Separation revival rate depends on separation sensitivity and is affected by many factors, including whether the patient is treated with antibiotics, the site and quantity of samples, the method and process of sample collection and processing, transport medium, separation medium and separation and culture conditions, etc. Isolation culture sensitivity varies widely (60–90%). The efficacy of the isolation medium in inhibiting the growth of bacteria in the gastrointestinal mucosa also affects the recovery rate of Helicobacter pylori. Although there are many media for culturing Helicobacter pylori, no rapid and sensitive isolation method has been reported.
  • Helicobacter pylori was isolated from gastric mucosa tissue samples using Columbia agar horse blood medium for 2-10 days, and GC agar medium was used to isolate Helicobacter pylori for 4-10 days.
  • the essence of rapid isolation and culture is to rapidly grow culturable live cell specimens containing Helicobacter pylori on a suitable medium to form Helicobacter pylori colonies.
  • the influencing factors of Helicobacter pylori isolation and culture are closely related to the isolation medium. Another important problem is that after the initial isolation of Helicobacter pylori, the subculture failed, so that the drug susceptibility test could not be completed.
  • the purpose of the present invention is to provide a method for forming a colony of Helicobacter pylori sensitive and rapid culture, which has the same good performance as initial isolation and subculture.
  • Another object of the present invention is to provide a method for evaluating the efficiency of Helicobacter pylori culture, and to provide an evaluation index for its efficiency.
  • the Helicobacter pylori sensitive and rapid culture colony formation method comprises the following steps:
  • Step 1 Open the Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, and preservation kit (produced by the Hainan Provincial Clinical Microbiology Testing and Research Center), take out the Helicobacter pylori rapid culture plate, vacuum-sealed packaging, and Store in waterproof and dark conditions;
  • Step 2 Take 2 zirconium beads with a particle size of 3-6 mm and put them into a 1-2 mL EP tube for sterilization;
  • Step 3 Open the Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, and preservation kit, take 0.5-1.0 mL of Helicobacter pylori preservation medium, put into the above-mentioned sterilized EP tube;
  • Step 4 Suspected Helicobacter pylori infection requires no treatment with PPI and antibiotics for at least 20 days, and gastrointestinal tissue sampling is performed. , stored at 2-8°C, the shelf life is not more than 10 days;
  • endoscope for gastrointestinal tissue sampling. If there is abnormality in gastrointestinal tissue (ulcer, erosion or mass, etc.), priority should be given to sampling at the junction of normal tissue and abnormal tissue;
  • Step 5 Grind the Helicobacter pylori storage medium containing the tissue sample with a tissue grinder 1-2 min;
  • Step 6 Open the vacuum-sealed package, take out 1 Helicobacter pylori rapid culture plate, draw 100-200 ⁇ L of ground samples and inoculate it on the Helicobacter pylori rapid culture plate, inoculate under sterile conditions, and inoculate the local
  • the temperature is lower than 40 °C; under the conditions of microaerobic and 100% humidity, cultivate at 35-37 °C for 48-120 hours, observe the colony morphology, the typical colony morphology is purple, round colony or diffuse colony morphology may appear;
  • Step 7 Freeze the Helicobacter pylori liquid sample at -70 ⁇ -80°C, draw 100-200 ⁇ L of the sample after thawing, and carry out resurrection culture according to the method of step 6;
  • Step 8 The Helicobacter pylori colony is prepared with sterile physiological saline to prepare 1.0 McFarland turbidity bacteria solution, and serially diluted to the 13th gradient or more; Scribe the center of its back and divide it into 2 areas equally; take 20 ⁇ L of diluted bacterial solution (such as 16 gradient) and inoculate them in the area of the streaking medium on the plate, and use a sterile inoculating loop to carry out continuous and dense circle coating; then follow the steps The method of 6 was cultured for 48-96 h, and the growth of a single colony on the medium plate was observed;
  • Step 9 Open the vacuum-sealed package, take out 1 Helicobacter pylori rapid culture plate, draw a cross in the center of its back, and divide it into 4 or 8 areas equally.
  • microaerophilic conditions refer to: 3-6% O 2 , 5-10% CO 2 , 5-10% H 2 and 76-86% N 2 .
  • the temperature condition of the culture is: 37°C
  • the microaerobic conditions are: 5% O 2 , 10% CO 2 , 5% H 2 and 80% N 2 , or 5% O 2 , 7.6% CO 2 , 7.6% H2 and 79.8% N2 .
  • the culture plate is vacuum-sealed and packaged to meet waterproof and light-proof conditions.
  • the local temperature for inoculation under sterile conditions is lower than 40°C.
  • the preservation medium contains zirconium beads.
  • the specimen is put into an EP tube containing zirconium beads as soon as possible, completely immersed in the medium, and the bottle cap is tightly closed, and then stored at 2-8°C.
  • the Helicobacter pylori colony is prepared with a physiological saline solution with a turbidity of 1.0 McFarland, and serially diluted to the 13th gradient or more.
  • a single colony was subcultured with a sterile inoculation loop to scrape a single colony in an area on the plate for continuous intensive circle coating and microaerophilic culture for 24 h.
  • the specimen is from the digestive system (such as gastrointestinal tissue, feces or saliva, etc.) of a person infected with Helicobacter pylori, and the infected person has not been treated with PPI and antibiotics for at least 20 days, and an endoscope is used to obtain gastrointestinal tissue samples , If there is abnormality in gastrointestinal tissue (inflammation, ulcer, erosion or mass, etc.), sampling should be prioritized at the junction of normal tissue and abnormal tissue.
  • the digestive system such as gastrointestinal tissue, feces or saliva, etc.
  • the present invention provides a Helicobacter pylori culture efficiency evaluation method, wherein the Helicobacter pylori culture efficiency evaluation indicators include, but are not limited to, the bacterial inhibition rate, the isolation rate, the survival rate of tissue strain passage, the passage resurrection rate of cryopreserved strains, the room temperature The survival rate of colony passage, the sensitivity of colony growth, the ratio of the number of colonies at different incubation times, the growth of single colony passaged cells and the growth of mixed colony cells were preserved.
  • the evaluation method includes:
  • Antibacterial rate (%) 1 - colony growth rate
  • Colony growth rate (%) CN/(CN1+CN2+CN3) ⁇ 100%
  • Antibacterial rate ⁇ 99% indicates high culture efficiency.
  • the evaluation method includes:
  • Each sample was inoculated in parallel with two different Helicobacter pylori isolation plates: GC agar medium plate and Columbia SBA plate; the two plates were cultured under the same conditions, and the culture time was 3-10 days.
  • the number of Helicobacter pylori positive cases obtained from the plate is N1
  • the number of Helicobacter pylori positive cases obtained by using the second separation plate is N2
  • the number of Helicobacter pylori positive cases obtained by combining the two separation plates is N3, which is calculated according to the following formula:
  • the separation rate of the second separation plate (%) N2/N3 ⁇ 100%
  • the plate separation rate of 100% indicates that the separation effect of Helicobacter pylori is good.
  • the isolation rate of Helicobacter pylori rapid culture plate was 100%.
  • the evaluation method includes: using plate separation to obtain the number of Helicobacter pylori strains as SN1, subculture each strain according to step 9 to obtain the number of Helicobacter pylori strains as SN2, and according to The following formula calculates the passage survival rate of tissue isolates:
  • the 100% survival rate of the strains isolated from the tissue means that the survival effect of the strains is good.
  • the survival rate of Helicobacter pylori rapid culture plate tissue was 100%.
  • the evaluation method includes: the number of samples FS1 of different strains that have been cryopreserved for at least 1 year at -70°C is operated according to the methods of steps 7 and 8, and the number of successful passaging and resurrection strains is FS2. , according to the following formula to calculate the passage resurrection rate of cryopreserved strains:
  • the evaluation method includes:
  • a 100% survival rate of colonies stored at room temperature means that the strains of Helicobacter pylori stored at room temperature for 24 h or more did not die.
  • the evaluation method includes: Passing the Helicobacter pylori type strain ATCC43504 according to the method in step 8, culturing for 72 h, calculating the number of colonies grown on the two plates, and the number of colonies cannot be less than 20 cfu.
  • the evaluation method includes:
  • the evaluation method includes: culturing the Helicobacter pylori type strain ATCC43504 according to the method in step 8, culturing the colony for 72 hours, and randomly selecting 16 single colonies to inoculate according to the method in step 9.
  • the evaluation method includes: culturing the Helicobacter pylori type strain ATCC43504 according to the method in step 8, culturing the colonies for 72 hours, randomly selecting 20 colonies, and scraping them with a moist sterile cotton swab respectively. Take 20 colonies and suspend them in a sterile tube containing 1.3 mL of sterile normal saline, and shake them with a shaker for 1-2 min, mix thoroughly, and measure the bacterial turbidity with a turbidimeter; if the bacterial turbidity is ⁇ 1.0 McFarland units, the plate meets the requirements for good growth of Helicobacter pylori colonies.
  • Helicobacter pylori rapid culture plate Liofilchem sheep blood medium plate and egg yolk medium plate were used to isolate, culture and passage Helicobacter pylori respectively. The results are shown in Table 1.
  • the present invention Compared with the prior art, the present invention has at least the following advantages: the present invention provides a method for forming a colony of Helicobacter pylori sensitive and rapid culture, and the method has the same good performance as primary isolation and subculture.
  • the invention provides a powerful tool for the rapid and efficient separation and subculture of Helicobacter pylori, and simultaneously provides an efficiency evaluation index for the Helicobacter pylori culture method.
  • Helicobacter pylori rapid isolation, culture, identification, drug susceptibility, and storage kits were provided by the Hainan Provincial Clinical Microbiology Testing and Research Center.
  • Liofilchem Sheep Blood Medium Plate Liofilchem, Italy.
  • Liofilchem Yolk Medium Plate Liofilchem, Italy.
  • Step 1 Open the Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, and preservation kit (produced by the Hainan Provincial Clinical Microbiology Testing and Research Center), take out the Helicobacter pylori rapid culture plate, vacuum-sealed packaging, and Store in waterproof and dark conditions.
  • Step 2 Take 2 zirconium beads with a particle size of 3-6 mm and put them into a 1.5 mL EP tube for sterilization.
  • Step 3 Open the Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, and preservation kit, take out 1.0 mL of Helicobacter pylori preservation medium, and put it into an EP tube containing 2 zirconium beads.
  • Step 4 Patients with suspected Helicobacter pylori infection are required to have not been treated with PPIs and antibiotics for at least 20 days.
  • Step 5 Grind the Helicobacter pylori preservation medium containing gastrointestinal mucosal tissue or other biological tissue with a tissue grinder for 2 min.
  • Step 6 Open the waterproof, light-proof, vacuum-sealed package, take out 1 Helicobacter pylori rapid culture plate (Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, preservation kit), and draw 100 ⁇ L of ground specimens Inoculate on Helicobacter pylori rapid culture plate, inoculate under sterile conditions, and inoculate the local temperature below 40 °C. Under microaerophilic conditions (5% O 2 , 7.6% CO 2 , 7.6% H 2 , 79.8% N 2 ) and humidity of 100%, culture at 37°C for 48-120 h, and observe the colony morphology. The typical colony morphology is purple. , round colonies or diffuse colonies may appear.
  • Helicobacter pylori rapid culture plate Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, preservation kit
  • 100 ⁇ L of ground specimens Inoculate on Helicobacter pylori rapid culture plate, inoculate under
  • Step 7 The liquid samples of Helicobacter pylori were frozen and stored at -80°C. After thawing, 100 ⁇ L of samples were drawn, and the method of step 6 was followed for resurrection culture.
  • Step 8 single colony growth method of strains: cryopreserved strains according to step 7 (clinical strains according to step 6), passage Helicobacter pylori strains for 18 hours, and prepare Helicobacter pylori (ATCC43504) colonies with physiological saline to prepare 1.0 turbidity bacteria solution, serially diluted to the 16th gradient. Open the vacuum-sealed bag, take out 2 Helicobacter pylori rapid culture plates, draw a line on the center of the back, and divide them into 2 areas equally. Inoculate 16 gradients of 20 ⁇ L of the diluted bacterial solution in the streak medium area of the plate respectively, and use a sterile inoculating loop to carry out continuous and dense circle coating. Incubate for 48-96 h according to the method in step 6, and observe the growth of purple single colonies on the medium plate.
  • Step 9 Single colony passaging method: Open the waterproof, light-proof, vacuum-sealed package, take out 1 Helicobacter pylori rapid culture plate (Helicobacter pylori rapid isolation and culture, identification, drug sensitivity, preservation kit), Draw a cross in the center of its back and divide it into 4 equal areas. Scrape a single colony with a sterile loop and apply a continuous, dense circle (about 17 mm in diameter) to an area on the plate. Each plate can be purified 4 strains of bacteria aseptically inoculated, and the local temperature of inoculation is lower than 40 °C. Under microaerophilic conditions (5% O 2 , 7.6% CO 2 , 7.6% H 2 , 79.8% N 2 ) and humidity of 100%, the cells were cultured at 37°C for 24 h, and the colony morphology was observed.
  • Helicobacter pylori rapid culture plate Helicobacter pylori rapid isolation and culture, identification, drug sensitivity, preservation kit
  • Identification Use the Helicobacter pylori rapid isolation and culture, identification, drug susceptibility, preservation kit (produced by the Hainan Provincial Clinical Microbiology Testing and Research Center) and antigen detection reagents, Gram staining of the colonies in steps 6 and 9, oxidase , catalase and urease test and antigen detection.
  • the identification results were consistent with the biological characteristics of Helicobacter pylori.
  • Helicobacter pylori antigen detection reagent (latex method), purchased from Aibo Biopharmaceutical (Hangzhou) Co., Ltd.
  • the evaluation indicators for the efficiency of Helicobacter pylori sensitive culture used in this example include: bacterial inhibition rate, isolation rate, tissue strain passage survival rate, cryopreservation strain passage passage resurrection rate, room temperature storage colony passage survival rate, colony growth sensitivity, different culture Time colony number ratio, single colony passage cell growth, mixed colony cell growth.
  • Antibacterial rate (%) 1 - colony growth rate
  • Colony growth rate (%) CN/(CN1+CN2+CN3) ⁇ 100%
  • Antibacterial rate ⁇ 99% indicates high culture efficiency.
  • the antibacterial rate of Helicobacter pylori rapid culture plate was 100%.
  • each sample was inoculated in parallel with two different Helicobacter pylori isolation plates: Helicobacter pylori rapid culture plate and Columbia SBA plate. identification by conventional methods.
  • the number of Helicobacter pylori positive cases obtained by using the first separation plate is N1
  • the number of Helicobacter pylori positive cases obtained by using the second separation plate is N2
  • the number of Helicobacter pylori positive cases obtained by combining the two plates is N3, calculated according to the following formula:
  • the separation rate of the second separation plate (%) N2/N3 ⁇ 100%
  • the plate separation rate of 100% indicates that the separation effect of Helicobacter pylori is good.
  • the isolation rate of Helicobacter pylori rapid culture plate was 100%.
  • the number of Helicobacter pylori strains obtained by using the plate is SN1, and each strain is subcultured according to step 8 to obtain Helicobacter pylori SN2, and the subculture survival rate of the tissue isolated strains is calculated according to the following formula:
  • the 100% survival rate of the strains isolated from the tissue means that the survival effect of the strains is good.
  • the survival rate of Helicobacter pylori rapid culture plate tissue was 100%.
  • the number FS1 of different strains preserved in -70°C for 1 year is implemented according to steps 7 and 8, and the number of successfully passaged and resurrected strains is FS2, and the passage and resurrection rate of cryopreserved strains is calculated according to the following formula:
  • Example 1 M plates of different strains of Helicobacter pylori were placed in a clean and humid container, and stored at room temperature of 25°C for 24 hours or more.
  • the survival rate of colony passage at room temperature was 100%, indicating that the strain of Helicobacter pylori stored at room temperature for 24 h did not die.
  • the survival rate of colony passage of Helicobacter pylori rapid culture plates stored at room temperature for 24 h was 100%.
  • the Helicobacter pylori type strain (ATCC43504) was passaged in step 8 and cultured for 72 hours, and the number of colonies grown on two plates was 36 cfu.
  • step 8 the colony of Helicobacter pylori type strain (ATCC43504) was cultured for 72 hours, and 16 single colonies were randomly selected and completely inoculated on 4 plates for 24 hours according to step 9.
  • 16 sterile tubes each tube of 1.3 mL of physiological saline, scrape 16 colonies with a moist sterile cotton swab and completely suspend them in the physiological saline of the 16 sterile tubes.
  • Use a turbidimeter to measure the bacterial turbidity of each tube. If there are 13 tubes with a turbidity of ⁇ 1.0 McFarland units, the plate meets the requirements for the rapid growth of Helicobacter pylori.
  • the growth of single colony passaged cells on the Helicobacter pylori rapid culture plate were: 4.0, 4.0, 4.0, 4.0, 4.0, 4.0, 3.0, 4.0, 3.2, 3.9, 2.8, 4.0, 1.3 and 3.2.
  • step 8 the colony of Helicobacter pylori type strain (ATCC43504) was cultured for 72 hours, 20 colonies were randomly selected, and 20 colonies were scraped with a moist sterile cotton swab and completely suspended in a sterile tube containing 1.3 mL of normal saline.
  • the shaker was shaken for 1 min, mixed well, and the bacterial turbidity was measured with a turbidimeter. If the bacterial turbidity is ⁇ 1.0 McFarland units, the plate meets the requirements for good growth of Helicobacter pylori colonies.
  • Helicobacter pylori rapid culture plate mixed colony cell growth amount was 1.1.

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Abstract

提供了一种幽门螺杆菌(Helicobacter pylori)敏感、快速培养菌落形成方法及其效能评价方法。菌落形成方法包括初次分离、冷冻保存复活、单个菌落生长及传代。效能评价方法为幽门螺杆菌培养方法提供效能评价指标。

Description

幽门螺杆菌敏感、快速培养菌落形成及其效能评价方法 技术领域
本发明涉及微生物培养技术，具体地说，涉及幽门螺杆菌敏感、快速培养菌落形成及其效能评价方法。
 背景技术
幽门螺杆菌（ Helicobacter pylori）是微需氧、能在人类胃肠粘膜持续定植并引起各种胃炎、十二指肠球炎和消化性溃疡最常见的致病菌，也和胃癌、胃MALT淋巴瘤密切相关，幽门螺杆菌感染率可达50%以上；并且和胃肠外感染疾病也有相关，如肺炎、菌血症、血清糖化血红蛋白水平相关等。因此诊断幽门螺杆菌感染很有必要。诊断幽门螺杆菌感染有多种技术，分为过筛试验和确诊试验。过筛试验包括脲酶试验、病理组织形态学检查、UBT试验、血清抗体、粪便抗原和分子生物学技术等；确诊试验只有幽门螺杆菌分离培养和免疫组化技术，幽门螺杆菌分离培养是所有诊断技术最特异的方法。常规病理形态学诊断并不能排除和幽门螺杆菌相似的弯曲菌和其它螺杆菌定植或感染。由于胃内有脲酶阳性的其它细菌存在，UBT和脲酶试验会产生假阳性结果，血清抗体可能反映继往感染，粪便抗原敏感性较差，分子生物学技术要求高，并且这些技术都不能提供可靠的药敏试验结果。幽门螺杆菌分离培养是这些诊断方法的“金标准”，并且是表型药敏试验和基因分型的必须过程。为了预防和治疗幽门螺杆菌感染引起的疾病，进行幽门螺杆菌快速分离培养有重要意义。培养分为初次分离（primary isolation）和传代培养（subculture）。菌落形成包括从人体标本初次分离菌落形成和传代培养的菌落形成。初次分离是将包含幽门螺杆菌活细胞的组织标本接种在合适的分离培养基（primary culture）并使其细胞生长尽可能多的菌落。传代培养是将幽门螺杆菌培养物接种在传代培养基并使其良好生长。分离培养基和传代培养基应该不同，组织标本通常含有除幽门螺杆菌之外的杂菌，需要在分离培养基添加抑制杂菌生长的抗生素而不影响幽门螺杆菌生长。传代培养基不需要添加抗生素，其他成分也可以与分离培养基不同，主要用于幽门螺杆菌生物学特征分析、表型药敏试验等试验。传代培养包括在平板培养基上菌落和冷冻保存培养物复活的菌落生长。传代培养复活率受培养基成分、稳定性等因素影响。影响幽门螺杆菌初次分离结果有二个重要因素：分离复活率（Recovery rates）和分离时间。分离复活率决定于分离敏感性，受很多因素影响，包括患者是否使用抗生素等治疗、取材部位和数量、取材和处理标本方式及过程、运送培养基、分离培养基和分离培养条件等，致使初次分离培养敏感性变化较大（60–90%）。分离培养基抑制胃肠粘膜杂菌生长的效能，也会影响幽门螺杆菌分离复活率。尽管培养幽门螺杆菌有很多培养基，但快速、敏感的分离方法还未见报道。应用Columbia琼脂马血培养基从胃粘膜组织标本分离幽门螺杆菌 2-10 天，应用GC琼脂培养基分离幽门螺杆菌4-10 天。快速分离培养的本质就是将含有幽门螺杆菌可培养的活细胞标本在合适的培养基上快速生长形成幽门螺杆菌菌落。幽门螺杆菌分离培养的影响因素和分离培养基密切相关。另一个重要问题是，幽门螺杆菌初次分离成功后，但传代培养失败，致使药敏试验等无法完成。有文献报道传代培养存活率（药敏完成率）只有84.8%，这说明幽门螺杆菌传代培养方法也存在重大缺陷。关于应用初次分离培养基作为传代培养基进行培养，从而敏感、快速形成幽门螺杆菌菌落还没有文献报道，对于幽门螺杆菌敏感、快速分离培养菌落形成方法也没有效能评价方法，培养基效能应满足公式：初次分离培养基=传代培养基。
技术问题
本发明目的在于提供一种幽门螺杆菌敏感、快速培养菌落形成方法，该方法具有初次分离和传代培养相同的良好效能。
本发明的另一目的是提供幽门螺杆菌培养效能评价方法，提供其效能评价指标。
 技术解决方案
为了实现本发明目的，第一方面，本发明提供的幽门螺杆菌敏感、快速培养菌落形成方法，包括以下步骤：
I、初次分离
步骤1：打开谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒（海南省临床微生物学检测与研究中心生产），取出谷氏幽门螺杆菌快速培养平板，进行真空密封包装，并在防水、避光条件下保存；
步骤2：取2枚粒径为3-6 mm的锆珠，放入1-2 mL EP管内，进行灭菌；
步骤3：打开谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒，取0.5-1.0 mL谷氏幽门螺杆菌保存培养基，放入上述灭菌的EP管内；
步骤4：疑为幽门螺杆菌感染者要求至少20天内未使用PPI和抗生素进行治疗，进行胃肠组织取样，将组织样本放入步骤3的EP管内，完全浸入培养基内，盖紧瓶盖后，放入2-8℃保存，保存期不超过10天；
使用内窥镜进行胃肠组织取样，如果胃肠组织存在异常（溃疡、糜烂或肿块等）须在正常组织与异常组织交界处优先取样；
步骤5：将含有组织样本的谷氏幽门螺杆菌保存培养基用组织研磨器进行研磨1-2 min；
步骤6：打开真空密封包装，取出1块谷氏幽门螺杆菌快速培养平板，吸取100-200 μL研磨的标本接种在谷氏幽门螺杆菌快速培养平板上，在无菌条件下接种，接种的局部温度低于40℃；在微需氧和湿度100%的条件下，35-37℃培养48-120 h，观察菌落形态，典型菌落形态为紫色、圆形菌落或可出现弥漫菌落形态；
II、冷冻保存复活
步骤7：将幽门螺杆菌液态样本于-70~-80℃冷冻保存，解冻后吸取100-200 μL样本，并按照步骤6的方法进行复活培养；
III、单个菌落生长
步骤8：将幽门螺杆菌菌落用无菌生理盐水调配1.0麦氏浊度菌液，连续倍比稀释至第13梯度以上；打开真空密封包装，取出2块谷氏幽门螺杆菌快速培养平板，在其背面中心划线，均等分成2个区域；取稀释菌液（如16梯度）20 μL分别接种在平板划线培养基区域内，用无菌接种环进行连续密集划圆涂布；然后按照步骤6的方法培养48-96 h，观察培养基平板上的单个菌落生长情况；
III、单个菌落传代
步骤9：打开真空密封包装，取出1块谷氏幽门螺杆菌快速培养平板，在其背面中心划十字线，均等分成4个或8个区域。用无菌接种环刮取单个菌落在平板上一个区域内进行连续密集划圆涂布，直径13-17 mm；每块平板可以纯化4株菌或8株菌；无菌接种，接种的局部温度低于40℃；在微需氧和湿度100%的条件下，35-37℃培养24 h，观察菌落形态。
本发明中，微需氧条件是指：3-6% O 2、5-10% CO 2、5-10% H 2和76-86% N 2。
优选地，培养的温度条件为：37℃，微需氧条件为：5% O 2、10% CO 2、5% H 2和80% N 2，或者，5% O 2、7.6% CO 2、7.6% H 2和79.8% N 2。
前述的方法，对培养平板进行真空密封包装，满足防水、避光条件。
前述的方法，在无菌条件接种的局部温度低于40℃。
前述的方法，保存培养基内含有锆珠。
前述的方法，标本尽快放入含有锆珠的EP管内，完全浸入培养基内，盖紧瓶盖后，放入2-8℃保存。
前述的方法，含有锆珠的EP管标本，进行研磨1-2 min。
前述的方法，单个菌落生长是将幽门螺杆菌菌落用生理盐水调配1.0麦氏浊度菌液，连续倍比稀释至第13梯度以上。
前述的方法，单个菌落传代用无菌接种环刮取单个菌落在平板上一个区域内进行连续密集划圆涂布微需氧培养24 h。
本发明中，所述标本来自幽门螺杆菌感染者的消化系统（如胃肠组织、粪便或唾液等），且感染者至少20天内没有使用PPI和抗生素治疗，使用内窥镜进行胃肠组织取材，如果胃肠组织存在异常（炎症、溃疡、糜烂或肿块等）须在正常组织与异常组织交界处优先取样。
第二方面，本发明提供幽门螺杆菌培养效能评价方法，其中，幽门螺杆菌培养效能评价指标包括但不限于抑杂菌率、分离率、组织菌株传代存活率、冷冻保存菌株传代复活率、室温保存菌落传代存活率、菌落生长灵敏度、不同培养时间菌落数量比、单个菌落传代细胞生长量以及混合菌落细胞生长量。
培养基效能应满足公式：初次分离培养基=传代培养基。
进一步地，当评价指标为抑杂菌率时，评价方法包括：
1）在血琼脂平板上分别接种大肠埃希氏菌ATCC25922和金黄葡萄球菌ATCC 25923，37℃培养16-18 h，将培养好的大肠埃希氏菌ATCC25922、金黄葡萄球菌ATCC 25923用无菌生理盐水分别配制1.0麦氏浊度菌液，连续倍比稀释至17梯度，取10 μL稀释后的菌液分别接种于血琼脂平板上，37℃培养24 h，在平板上形成的菌落数分别记为CN1、CN2；
2）在沙保弱氏培养基平板上接种白色念珠菌ATCC 90028，37℃培养16-18 h，将培养好的白色念珠菌ATCC 90028用无菌生理盐水配制1.0麦氏浊度菌液，连续倍比稀释至11梯度，取10 μL稀释后的菌液接种于沙保弱氏培养基平板上，37℃培养24 h，在平板上形成的菌落数记为CN3；
3）在分离平板的背面中心划Y字线，将平板均等分成3个区域，在3个区域分别接种10 μL稀释后的大肠埃希氏菌ATCC25922菌液、金黄葡萄球菌ATCC 25923菌液和白色念珠菌ATCC 90028菌液，然后在3-6% O 2、5-10% CO 2、5-10% H 2和76-86% N 2的微需氧条件以及温度35-37℃、湿度100%的条件下培养72 h，将3个区域的菌落总数记为CN；
4）按照以下公式计算抑杂菌率：
抑杂菌率（%）=1-菌落生长率
菌落生长率（%）= CN/（CN1+CN2+CN3）×100%
抑杂菌率≥99%表示培养效能高。
进一步地，当评价指标为菌株分离率时，评价方法包括：
每个样本分别平行接种两种不同的幽门螺杆菌分离平板：GC琼脂培养基平板和columbia SBA平板；两种平板培养条件相同，培养时间3-10天，按常规方法进行鉴定；使用第一分离平板得到幽门螺杆菌阳性例数为N1，使用第二分离平板得到幽门螺杆菌阳性例数为N2，综合两种分离平板得到幽门螺杆菌阳性例数为N3，按照以下公式计算：
第一分离平板分离率（%）=N1/N3×100%
第二分离平板分离率（%）=N2/N3×100%
平板分离率100%表示幽门螺杆菌分离效果好。谷氏幽门螺杆菌快速培养平板分离率为100%。
进一步地，当评价指标为组织分离菌株传代存活率时，评价方法包括：采用平板分离得到幽门螺杆菌菌株数为SN1，将每株菌按照步骤9传代培养得到幽门螺杆菌菌株数为SN2，按照以下公式计算组织分离菌株传代存活率：
组织分离菌株传代存活率（%）=SN2/SN1×100%
组织分离菌株传代存活率100%表示菌株传代存活效果好。谷氏幽门螺杆菌快速培养平板组织分离菌株传代存活率100%。
进一步地，当评价指标为冷冻保存菌株传代复活率时，评价方法包括：将-70℃冷冻保存至少1年的不同菌株样本数FS1按照步骤7、8的方法操作，传代复活成功菌株数为FS2，按照以下公式计算冷冻保存菌株传代复活率：
冷冻保存菌株传代复活率（%）=FS2/FS1×100%
冷冻保存菌株传代复活率100%表示幽门螺杆菌保存和复活效果好。
进一步地，当评价指标为室温保存菌落传代存活率时，评价方法包括：
按照步骤1-9的方法操作，每块平板纯化4株菌；将M块平板幽门螺杆菌不同菌株放在洁净、湿润的容器中，室温25℃存放24 h及以上；各菌株按照步骤9方法接种培养，菌株传代存活数为VS，按照以下公式计算室温保存菌落传代存活率：
室温保存菌落传代存活率（%）= VS/4M×100%
室温保存菌落传代存活率100%表示幽门螺杆菌室温保存24 h及以上菌株没有死亡。
进一步地，当评价指标为菌落生长灵敏度时，评价方法包括：按照步骤8的方法传代幽门螺杆菌模式菌株ATCC43504，培养72 h，计算两块平板生长菌落数，菌落数不能低于20 cfu。
进一步地，当评价指标为不同培养时间菌落数量比时，评价方法包括：
1）按照步骤8的方法培养48 h，形成的菌落数记为NC2；
2）将上述平板继续培养72 h，形成的菌落数记为NC3；
3）将上述平板继续培养96 h，形成的菌落数记为NC4；
4）如果同时满足以下两个条件：①NC2/NC3×100%.≥80%，②NC3/NC4×100%≥90%，表示幽门螺杆菌在该培养基平板上48h形成肉眼可见菌落，达到了快速生长的效能，弥漫生长菌落除外。
进一步地，当评价指标为单个菌落传代细胞生长量时，评价方法包括：按照步骤8的方法培养幽门螺杆菌模式菌株ATCC43504，菌落培养72 h，随机选取16个单菌落按照步骤9的方法分别接种在4块平板培养24h；取16只无菌管，每管装有1.3 mL无菌生理盐水，用湿润的无菌棉签分别刮取16个菌落悬浮于16只无菌管的生理盐水中；用比浊仪测定每管细菌浊度，如果其中有13只管浊度≥1.0麦氏单位，则该平板符合幽门螺杆菌快速生长要求。
进一步地，当评价指标为混合菌落细胞生长量时，评价方法包括：按照步骤8的方法培养幽门螺杆菌模式菌株ATCC43504，菌落培养72 h，随机选取20个菌落，用湿润的无菌棉签分别刮取20个菌落悬浮于1只含有1.3 mL无菌生理盐水的无菌管中，震荡仪震荡1-2 min，充分混匀，用比浊仪测定其细菌浊度；如果细菌浊度≥1.0麦氏单位，则该平板符合幽门螺杆菌菌落良好生长要求。
分别使用谷氏幽门螺杆菌快速培养平板、Liofilchem绵羊血培养基平板与卵黄培养基平板进行幽门螺杆菌的分离、培养及传代，结果见表1。
表1
  谷氏幽门螺杆菌快速培养平板 Liofilchem绵羊血培养基平板 Liofilchem卵黄培养基平板
胃活检标本（n=146）      
幽门螺杆菌培养阳性      
2天 26 （37.5%） 0 0
3天 44 （62.5%） 43 （95.6%） 22 （88%）
5天 0 2 （4.4%） 3 （12%）
传代存活率      
1~2天 100% — —
2~5天 — 51/69（73.91%） 23/29（79.31%）
分离率（%） 100% 64.3% 35.7%
阳性天数 2.0~3.0 3.0~5.0 3.0~5.0
每个平板菌落数量 3000~4000 500~1000 1000~2000
平板污染率 10% 13.3% 12%
有益效果
与现有技术相比，本发明至少具有以下优点：本发明提供一种幽门螺杆菌敏感、快速培养菌落形成方法，该方法具有初次分离和传代培养相同的良好效能。本发明为幽门螺杆菌的快速、高效地分离、传代培养提供有力工具，同时为幽门螺杆菌培养方法提供效能评价指标。
 本发明的实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中使用的培养基：
谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒，由海南省临床微生物学检测与研究中心提供。
沙保弱氏培养基：BD，France。
Columbia BLOOD AGAR：OXOID，ENGLAND。
GC琼脂培养基：BD，France。
Liofilchem绵羊血培养基平板：Liofilchem，Italy。
Liofilchem卵黄培养基平板：Liofilchem，Italy。
实施例 1 幽门螺杆菌敏感、快速培养菌落形成方法
包括以下步骤：
（一）初次分离方法
步骤1：打开谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒（海南省临床微生物学检测与研究中心生产），取出谷氏幽门螺杆菌快速培养平板，进行真空密封包装，并在防水、避光条件下保存。
步骤2：取2枚粒径为3-6 mm的锆珠，放入1.5 mL EP管内，进行灭菌。
步骤3：打开谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒，取出1.0 mL谷氏幽门螺杆菌保存培养基，放入含有2枚锆珠的EP管内。
步骤4：疑为幽门螺杆菌感染患者要求至少20天内没有使用PPI和抗生素治疗。使用内窥镜进行胃肠组织取材，如果胃肠组织存在异常（溃疡、糜烂或肿块等）须在正常组织与异常组织交界处优先取材。尽快放入步骤3的EP管内，完全浸入培养基内，瓶盖盖紧后，放入2-8℃保存，保存期不能超10天。
步骤5：将含有胃肠粘膜组织或其他生物学组织的谷氏幽门螺杆菌保存培养基用组织研磨器进行研磨2 min。
步骤6：打开防水、避光、真空密封包装，取出1块谷氏幽门螺杆菌快速培养平板（谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒），吸取100 μL研磨的标本接种在谷氏幽门螺杆菌快速培养平板上，在无菌条件下接种，接种的局部温度低于40℃。在微需氧（5% O 2、7.6% CO 2、7.6% H 2、79.8% N 2）和湿度为100%条件下，37℃培养48-120 h，观察菌落形态，典型菌落形态为紫色、圆形菌落或可出现弥漫菌落形态。
（二）冷冻保存复活、单个菌落生长方法及传代方法
步骤7（冷冻保存复活方法）：将幽门螺杆菌液态样本于-80℃冷冻保存，解冻后吸取100 μL样本，并按照步骤6的方法进行复活培养。
步骤8（菌株单个菌落生长方法）：冷冻保存菌株按步骤7（临床菌株按步骤6）传代幽门螺杆菌菌株培养18 h，将幽门螺杆菌（ATCC43504）菌落用生理盐水调配1.0麦氏浊度菌液，连续倍比稀释至第16梯度。打开真空密封包，取出2块谷氏幽门螺杆菌快速培养平板，在其背面中心划线，均等分成2个区域。将稀释菌液16梯度20 μL分别接种在平板划线培养基区域内，用无菌接种环进行连续密集划圆涂布。按步骤6方法培养48-96 h，观察培养基平板上的紫色单个菌落生长情况。
步骤9（单个菌落传代方法）：打开防水、避光、真空密封包装，取出1块谷氏幽门螺杆菌快速培养平板（谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒），在其背面中心划十字线，均等分成4个区域。用无菌接种环刮取单个菌落在平板上一个区域内进行连续密集划圆涂布（直径约17 mm）。每块平板可以纯化4株菌无菌接种，接种的局部温度低于40℃。在微需氧（5% O 2、7.6% CO 2、7.6% H 2、79.8% N 2）和湿度为100%条件下，37℃培养24 h，观察菌落形态。
鉴定：应用谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒（海南省临床微生物学检测与研究中心生产）及抗原检测试剂，对步骤6、9菌落进行革兰染色，氧化酶、触酶和脲酶试验及抗原检测。鉴定结果符合幽门螺杆菌生物学特征。幽门螺杆菌抗原检测试剂（乳胶法）、购自艾博生物医药（杭州）有限公司。
实施例 2 幽门螺杆菌培养效能评价方法
本实施例中采用的幽门螺杆菌敏感培养效能评价指标包括：抑杂菌率、分离率、组织菌株传代存活率、冷冻保存菌株传代复活率、室温保存菌落传代存活率、菌落生长灵敏度、不同培养时间菌落数量比、单个菌落传代细胞生长量、混合菌落细胞生长量。
1、抑杂菌率
1）在血琼脂平板上分别接种大肠埃希氏菌ATCC25922和金黄葡萄球菌ATCC 25923，37℃培养16-18 h，将培养好的大肠埃希氏菌ATCC25922、金黄葡萄球菌ATCC 25923用无菌生理盐水分别配制1.0麦氏浊度菌液，连续倍比稀释至17梯度，取10 μL稀释后的菌液分别接种于血琼脂平板上，37℃培养24 h，在平板上形成的菌落数分别记为CN1、CN2；
2）在沙保弱氏培养基平板上接种白色念珠菌ATCC 90028，37℃培养16-18 h，将培养好的白色念珠菌ATCC 90028用无菌生理盐水配制1.0麦氏浊度菌液，连续倍比稀释至11梯度，取10 μL稀释后的菌液接种于沙保弱氏培养基平板上，37℃培养24 h，在平板上形成的菌落数记为CN3；
3）在分离平板的背面中心划Y字线，将平板均等分成3个区域，在3个区域分别接种10 μL稀释后的大肠埃希氏菌ATCC25922菌液、金黄葡萄球菌ATCC 25923菌液和白色念珠菌ATCC 90028菌液，然后在5% O 2、7.6% CO 2、7.6% H 2和79.8% N 2的微需氧条件以及温度35-37℃、湿度100%的条件下培养72 h，将3个区域的菌落总数记为CN；
4）按照以下公式计算抑杂菌率：
抑杂菌率（%）=1-菌落生长率
菌落生长率（%）= CN/（CN1+CN2+CN3）×100%
抑杂菌率≥99%表示培养效能高。谷氏幽门螺杆菌快速培养平板抑杂菌率为100%。
2、分离率（Isolation rate）
按照实施例1方法，每个标本分别平行接种两种不同幽门螺杆菌分离平板：谷氏幽门螺杆菌快速培养平板和columbia SBA平板，两种平板培养条件相同，培养时间是3-10天，按常规方法进行鉴定。使用第一分离平板得到幽门螺杆菌阳性例数为N1，使用第二分离平板得到幽门螺杆菌阳性例数为N2，综合两种平板得到幽门螺杆菌阳性例数为N3，按照以下公式计算：
第一分离平板分离率（%）=N1/N3×100%
第二分离平板分离率（%）=N2/N3×100%
平板分离率100%表示幽门螺杆菌分离效果好。谷氏幽门螺杆菌快速培养平板分离率为100%。
3、组织分离菌株传代存活率
按照实施例1方法，使用平板得到幽门螺杆菌菌株数为SN1，将每株菌按步骤8传代培养得到幽门螺杆菌 SN2，按照以下公式计算组织分离菌株传代存活率：
组织分离菌株传代存活率（%）=SN2/SN1×100%
组织分离菌株传代存活率100%表示菌株传代存活效果好。谷氏幽门螺杆菌快速培养平板组织分离菌株传代存活率100%。
4、冷冻保存菌株传代复活率
按照实施例1方法，将-70℃冷冻保存1年的不同菌株保存标本数FS1按步骤7、8进行实施，传代复活成功菌株数为FS2，按照以下公式计算冷冻保存菌株传代复活率：
冷冻保存菌株传代复活率（%）=FS2/FS1×100%
冷冻保存菌株传代复活率100%表示幽门螺杆菌保存和复活效果好。谷氏幽门螺杆菌快速培养平板冷冻保存菌株传代复活率100%。
5、室温保存菌落传代存活率
按照实施例1方法，将M块平板幽门螺杆菌不同菌株放在洁净、湿润的容器中，室温25℃存放24 h及以上。幽门螺杆菌每株菌按照实施例2方法接种培养，菌株存活数为VS，室温保存菌落传代存活率=VS/4M。室温保存菌落传代存活率100%表示幽门螺杆菌室温保存24 h菌株没有死亡。谷氏幽门螺杆菌快速培养平板室温保存24 h菌落传代存活率100%。
6、菌落生长灵敏度
按照实施例2方法按步骤8传代幽门螺杆菌 模式菌株（ATCC43504）培养72h，两块平板生长菌落数36cfu。
7、不同培养时间生长菌落数量比
按步骤8方法培养48h，形成的菌落数记为NC2；将上述平板继续培养72h，形成的菌落数记为NC3；将上述平板继续培养96h，形成的菌落数记为NC4；如果同时满足以下两个条件：①NC2/NC3×100%.≥80%，②NC3/NC4×100%≥90%，表示幽门螺杆菌 在该培养基平板上48h形成肉眼可见菌落，达到了快速生长的效能。谷氏幽门螺杆菌快速培养平板NC2/NC3=87%，NC3/NC4=92%。
8、单个菌落传代细胞生长量
按步骤8实施，幽门螺杆菌模式菌株（ATCC43504）菌落培养72 h，随机选取16个单个菌落按步骤9实施分别完全接种在4块平板培养24 h。取16只无菌管，每管1.3 mL生理盐水，用湿润的无菌棉签分别刮取16个菌落完全悬浮于16只无菌管的生理盐水中。用比浊仪测定每管细菌浊度，如果其中有13只管浊度≥1.0麦氏单位，该平板符合幽门螺杆菌快速生长要求。谷氏幽门螺杆菌快速培养平板单个菌落传代细胞生长量分别为：4.0、4.0、4.0、4.0、4.0、4.0、4.0、4.0、3.0、4.0、3.2、3.9、2.8、4.0、1.3和3.2。
9、混合菌落细胞生长量
按步骤8实施，幽门螺杆菌模式菌株（ATCC43504）菌落培养72 h，随机选取20个菌落，用湿润的无菌棉签分别刮取20个菌落完全悬浮于1只含有1.3 mL生理盐水的无菌管中，震荡仪震荡1 min，充分混匀，用比浊仪测定其细菌浊度。如果细菌浊度≥1.0麦氏单位，该平板符合幽门螺杆菌菌落良好生长要求。谷氏幽门螺杆菌快速培养平板混合菌落细胞生长量为1.1。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (20)

  1. 幽门螺杆菌（ Helicobacter pylori）敏感、快速培养菌落形成方法，其特征在于，包括以下步骤：
    I、初次分离
    步骤1：打开谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒，取出谷氏幽门螺杆菌快速培养平板，进行真空密封包装，并在防水、避光条件下保存；
    步骤2：取2枚粒径为3-6 mm的锆珠，放入1-2 mL EP管内，进行灭菌；
    步骤3：打开谷氏幽门螺杆菌快速分离培养、鉴定、药敏、保存试剂盒，取0.5-1.0 mL谷氏幽门螺杆菌保存培养基，放入上述灭菌的EP管内；
    步骤4：疑为幽门螺杆菌感染者要求至少20天内未使用PPI和抗生素进行治疗，进行胃肠组织取样，将组织样本放入步骤3的EP管内，完全浸入培养基内，盖紧瓶盖后，放入2-8℃保存，保存期不超过10天；
    步骤5：将含有组织样本的谷氏幽门螺杆菌保存培养基用组织研磨器进行研磨1-2 min；
    步骤6：打开真空密封包装，取出1块谷氏幽门螺杆菌快速培养平板，吸取100-200 μL研磨的标本接种在谷氏幽门螺杆菌快速培养平板上，在无菌条件下接种，接种的局部温度低于40℃；在微需氧和湿度100%的条件下，35-37℃培养48-120 h，观察菌落形态，典型菌落形态为紫色、圆形菌落或可出现弥漫菌落形态；
    II、冷冻保存复活
    步骤7：将幽门螺杆菌液态样本于-70~-80℃冷冻保存，解冻后吸取100-200 μL样本，并按照步骤6的方法进行复活培养；
    III、单个菌落生长
    步骤8：将幽门螺杆菌菌落用无菌生理盐水调配1.0麦氏浊度菌液，连续倍比稀释至第13梯度以上；打开真空密封包装，取出2块谷氏幽门螺杆菌快速培养平板，在其背面中心划线，均等分成2个区域；取稀释菌液20 μL分别接种在平板划线培养基区域内，用无菌接种环进行连续密集划圆涂布；然后按照步骤6的方法培养48-96 h，观察培养基平板上的单个菌落生长情况；
    III、单个菌落传代
    步骤9：打开真空密封包装，取出1块谷氏幽门螺杆菌快速培养平板，在其背面中心划十字线，均等分成4个或8个区域；用无菌接种环刮取单个菌落在平板上一个区域内进行连续密集划圆涂布，直径13-17 mm；接种的局部温度低于40℃；在微需氧和湿度100%的条件下，35-37℃培养24 h，观察菌落形态；
    其中，微需氧条件是指：3-6% O 2、5-10% CO 2、5-10% H 2和76-86% N 2。
  2. 根据权利要求1所述的方法，其特征在于，对培养平板进行真空密封包装，满足防水、避光条件。
  3. 根据权利要求1所述的方法，其特征在于，培养的温度条件为：37℃，微需氧条件为：5% O 2、10% CO 2、5% H 2和80% N 2，或者，5% O 2、7.6% CO 2、7.6% H 2和79.8% N 2。
  4. 根据权利要求1所述的方法，其特征在于，在无菌条件接种的局部温度低于40℃。
  5. 根据权利要求1所述的方法，其特征在于，保存培养基内含有锆珠。
  6. 根据权利要求1所述的方法，其特征在于，标本尽快放入含有锆珠的EP管内，完全浸入培养基内，盖紧瓶盖后，放入2-8℃保存。
  7. 根据权利要求1-6所述的方法，其特征在于，含有锆珠的EP管标本，进行研磨1-2 min。
  8. 根据权利要求1所述的方法，其特征在于，单个菌落生长是将幽门螺杆菌菌落用生理盐水调配1.0麦氏浊度菌液，连续倍比稀释至第13梯度以上。
  9. 根据权利要求1所述的方法，其特征在于，单个菌落传代用无菌接种环刮取单个菌落在平板上一个区域内进行连续密集划圆涂布微需氧培养24 h。
  10. [根据细则26改正21.05.2021]
    根据权利要求1所述的方法，其特征在于，在胃肠组织存在异常的情况下，在正常组织与异常组织交界处进行取样；
    其中，胃肠组织存在异常包括炎症、溃疡、糜烂或肿块。
  11. [根据细则26改正21.05.2021]
    幽门螺杆菌培养效能评价方法，其特征在于，幽门螺杆菌培养效能评价指标包括抑杂菌率、菌株分离率、菌株传代存活率、冷冻保存菌株传代复活率、室温保存菌落传代存活率、菌落生长灵敏度、不同培养时间菌落数量比、单个菌落传代细胞生长量以及混合菌落细胞生长量。
  12. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为抑杂菌率时，评价方法包括：
    1）在血琼脂平板上分别接种大肠埃希氏菌ATCC25922和金黄葡萄球菌ATCC 25923，37℃培养16-18 h，将培养好的大肠埃希氏菌ATCC25922、金黄葡萄球菌ATCC 25923用无菌生理盐水分别配制1.0麦氏浊度菌液，连续倍比稀释至17梯度，取10 μL稀释后的菌液分别接种于血琼脂平板上，37℃培养24 h，在平板上形成的菌落数分别记为CN1、CN2；
    2）在沙保弱氏培养基平板上接种白色念珠菌ATCC 90028，37℃培养16-18 h，将培养好的白色念珠菌ATCC 90028用无菌生理盐水配制1.0麦氏浊度菌液，连续倍比稀释至11梯度，取10 μL稀释后的菌液接种于沙保弱氏培养基平板上，37℃培养24 h，在平板上形成的菌落数记为CN3；
    3）在分离平板的背面中心划Y字线，将平板均等分成3个区域，在3个区域分别接种10 μL稀释后的大肠埃希氏菌ATCC25922菌液、金黄葡萄球菌ATCC 25923菌液和白色念珠菌ATCC 90028菌液，然后在3-6% O 2、5-10% CO 2、5-10% H 2和76-86% N 2的微需氧条件以及温度35-37℃、湿度100%的条件下培养72 h，将3个区域的菌落总数记为CN；
    4）按照以下公式计算抑杂菌率：
    抑杂菌率（%）=1-菌落生长率
    菌落生长率（%）= CN/（CN1+CN2+CN3）×100%
    抑杂菌率≥99%表示培养效能高。
  13. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为菌株分离率时，评价方法包括：每个样本分别平行接种两种不同的幽门螺杆菌分离平板：GC琼脂培养基平板和columbia SBA平板；两种平板培养条件相同，培养时间3-10天，按常规方法进行鉴定；使用第一分离平板得到幽门螺杆菌阳性例数为N1，使用第二分离平板得到幽门螺杆菌阳性例数为N2，综合两种分离平板得到幽门螺杆菌阳性例数为N3，按照以下公式计算：
    第一分离平板分离率（%）=N1/N3×100%
    第二分离平板分离率（%）=N2/N3×100%
    平板分离率100%表示幽门螺杆菌分离效果好。
  14. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为组织分离菌株传代存活率时，评价方法包括：采用平板分离得到幽门螺杆菌菌株数为SN1，将每株菌按照步骤9传代培养得到幽门螺杆菌菌株数为SN2，按照以下公式计算组织分离菌株传代存活率：
    组织分离菌株传代存活率（%）=SN2/SN1×100%
    组织分离菌株传代存活率100%表示菌株传代存活效果好。
  15. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为冷冻保存菌株传代复活率时，评价方法包括：将-70℃冷冻保存至少1年的不同菌株样本数FS1按照步骤7、8的方法操作，传代复活成功菌株数为FS2，按照以下公式计算冷冻保存菌株传代复活率：
    冷冻保存菌株传代复活率（%）=FS2/FS1×100%
    冷冻保存菌株传代复活率100%表示幽门螺杆菌保存和复活效果好。
  16. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为室温保存菌落传代存活率时，评价方法包括：按照步骤1-9的方法操作，每块平板纯化4株菌；将M块平板幽门螺杆菌不同菌株放在洁净、湿润的容器中，室温25℃存放24 h及以上；各菌株按照步骤9方法接种培养，菌株传代存活数为VS，按照以下公式计算室温保存菌落传代存活率：
    室温保存菌落传代存活率（%）= VS/4M×100%
    室温保存菌落传代存活率100%表示幽门螺杆菌室温保存24 h及以上菌株没有死亡。
  17. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为菌落生长灵敏度时，评价方法包括：按照步骤8的方法传代幽门螺杆菌模式菌株ATCC43504，培养72 h，计算两块平板生长菌落数，菌落数不能低于20 cfu。
  18. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为不同培养时间菌落数量比时，评价方法包括：
    1）按照步骤8的方法培养48 h，形成的菌落数记为NC2；
    2）将上述平板继续培养72 h，形成的菌落数记为NC3；
    3）将上述平板继续培养96 h，形成的菌落数记为NC4；
    4）如果同时满足以下两个条件：①NC2/NC3×100%.≥80%，②NC3/NC4×100%≥90%，表示幽门螺杆菌在该培养基平板上48h形成肉眼可见菌落，达到了快速生长的效能，弥漫生长菌落除外。
  19. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为单个菌落传代细胞生长量时，评价方法包括：按照步骤8的方法培养幽门螺杆菌模式菌株ATCC43504，菌落培养72 h，随机选取16个单菌落按照步骤9的方法分别接种在4块平板培养24 h；取16只无菌管，每管装有1.3 mL无菌生理盐水，用湿润的无菌棉签分别刮取16个菌落悬浮于16只无菌管的生理盐水中；用比浊仪测定每管细菌浊度，如果其中有13只管浊度≥1.0麦氏单位，则该平板符合幽门螺杆菌快速生长要求。
  20. [根据细则26改正21.05.2021]
    根据权利要求11所述的方法，其特征在于，当评价指标为混合菌落细胞生长量时，评价方法包括：按照步骤8的方法培养幽门螺杆菌模式菌株ATCC43504，菌落培养72 h，随机选取20个菌落，用湿润的无菌棉签分别刮取20个菌落悬浮于1只含有1.3 mL无菌生理盐水的无菌管中，震荡仪震荡1-2 min，充分混匀，用比浊仪测定其细菌浊度；如果细菌浊度≥1.0麦氏单位，则该平板符合幽门螺杆菌菌落良好生长要求。
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