阅读说明：本技术 一种厌氧微生物的体外检测试剂盒 (In-vitro detection kit for anaerobic microorganisms ) 是由 娄斌 章晓庆 于 2021-01-27 设计创作，主要内容包括：本发明提供了一种厌氧微生物的体外检测试剂盒,涉及生物化学检测技术领域,该试剂盒包括培养液、吸附树脂、培养瓶瓶体、瓶盖和感受器；所述培养液包括胰蛋白胨、明胶蛋白胨、脑心浸液、L-精氨酸、L-半胱氨酸盐酸盐、活性增效调节剂、枸橼酸钠、磷酸二氢钾和维生素C。该试剂盒能够有效地中和抗生素,为微生物提供更有利的生长条件,进而提高厌氧微生物的阳性检出率并缩短检出时间。(The invention provides an in-vitro detection kit for anaerobic microorganisms, which relates to the technical field of biochemical detection and comprises a culture solution, adsorption resin, a culture bottle body, a bottle cap and a receptor; the culture solution comprises tryptone, gelatin peptone, brain heart extract, L-arginine, L-cysteine hydrochloride, activity synergistic regulator, sodium citrate, potassium dihydrogen phosphate and vitamin C. The kit can effectively neutralize antibiotics, provides more favorable growth conditions for microorganisms, further improves the positive detection rate of anaerobic microorganisms and shortens the detection time.)

1. An in vitro detection kit for anaerobic microorganisms, which is characterized in that: comprises a culture solution, adsorption resin, a culture bottle body, a bottle cap and a receptor;

the culture solution comprises tryptone, gelatin peptone, brain heart extract, L-arginine, L-cysteine hydrochloride, activity synergistic regulator, sodium citrate, potassium dihydrogen phosphate and vitamin C.

2. The in vitro test kit according to claim 1, characterized in that: the culture solution comprises: 1-10g/L tryptone, 1-10g/L gelatin peptone, 1-10g/L brain heart extract, 1-10g/L L-arginine, 1-10g/L L-cysteine hydrochloride, 0.08-0.3g/L activity synergistic regulator, 0.2-0.5g/L sodium citrate, 0.2-0.5g/L potassium dihydrogen phosphate and 0.8-2g/L vitamin C.

3. The in vitro test kit according to claim 1, characterized in that: the activity synergistic regulator comprises phytosterol and hemin.

4. The in vitro test kit according to claim 3, characterized in that: the weight ratio of the phytosterol to the hemin is 1-2: 3-5.

5. The in vitro test kit according to claim 3, characterized in that: the phytosterol comprises one or more of sitosterol, stigmasterol and campesterol.

6. The in vitro test kit according to claim 1, characterized in that: the adsorption resin is anion exchange resin and macroporous adsorption resin.

7. The in vitro test kit according to claim 1, characterized in that: the sensor is positioned at the bottom of the flask body and contains fluorescent dye.

8. The in vitro test kit according to claim 1, characterized in that: the culture solution is prepared by using double distilled water.

9. Use of a kit according to any one of claims 1 to 8 for the in vitro detection of anaerobic microorganisms for non-diagnostic purposes.

10. Use according to claim 9, characterized in that: the anaerobic microorganisms include Clostridium histolyticum, Bacteroides fragilis, Bacteroides vulgatus, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, and Clostridium perfringens.

Technical Field

The invention relates to the technical field of biochemical detection, in particular to an in-vitro detection kit for anaerobic microorganisms.

Background

Bacteremia refers to the condition that external bacteria enter the blood system through the body surface entrance or infected wound, and multiply in the blood of human body and spread throughout the body with the blood flow. Bacteremia is caused by bacteria entering from local focus, and no toxic symptom exists in the whole body, but bacteria can be detected in blood, and the bacteremia mainly occurs in the early stage of inflammation. 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. Since most of septicemia is secondary to various infections and lacks specific clinical manifestations, missed diagnosis or misdiagnosis is easy to occur. Diagnosis of the above conditions is often performed by blood culture methods. The blood culture is a culture method for determining pathogenic bacteria by inoculating a fresh isolated blood specimen on a nutrient medium, growing and propagating bacteria with high nutrient requirements under the conditions of certain temperature, humidity and the like, and identifying the bacteria.

Chinese patent CN104450506B discloses an anaerobic blood culture medium, a culture bottle and a preparation method of the culture bottle, wherein two substances, namely glucose oxidase and peroxidase, are newly added in the culture medium, and the substances can remove oxygen in the culture medium, and hydrogen peroxide and other cytotoxic substances which are generated in the metabolic process and are harmful to bacteria, so that the problems of low positive detection rate, complex production and high cost in the traditional method are solved. However, the invention mainly adopts components such as sodium polyanthranilate, glucose oxidase and peroxidase, so that the antibiotic is effectively neutralized, the influence of the antibiotic on a blood culture medium is reduced, and generally, the positive detection rate is still relatively low, so that the invention has certain limitation.

Chinese patent CN102925344A discloses an improved anaerobic two-phase blood culture bottle, the solid phase is prepared by mixing CDC anaerobic broth, agar powder, yeast extract powder, nucleic acid, sodium glutamate and vitamin C according to a certain weight ratio, and adding deionized water. The liquid phase is prepared by mixing CDC anaerobic broth, yeast extract powder, SPS, coenzyme I, vitamin B6, vitamin K3, vitamin B12, L-glutamine, L-cystine, L-cysteine hydrochloride, arginine, adenine sulfate, guanine hydrochloride, vitamin C and isovitamin C according to a certain weight ratio and adding deionized water. The reasonable formula proportion, the perfect nutrient components and the environment condition which is more suitable for the growth of the anaerobic bacteria are more beneficial to the growth of various anaerobic bacteria. And can greatly improve the positive detection rate and the detection time. However, the present invention does not specify the kind of anaerobic bacteria, and thus it is not known whether the blood culture bottle has universality for anaerobic bacteria detection, and the detection rate and detection time are not specified, so that it is difficult to determine the actual effect of the blood culture bottle.

Aiming at the problems of unclear effect, unclear universality and the like in the prior art, an in-vitro detection kit for anaerobic microorganisms is required to be found, so that the kit can be generally suitable for detection of various anaerobic bacteria, and has high detection rate and short detection time.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an in-vitro detection kit for anaerobic microorganisms, which can effectively neutralize antibiotics and provide more favorable growth conditions for the microorganisms, thereby improving the positive detection rate of the anaerobic microorganisms and shortening the detection time.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides an in-vitro detection kit for anaerobic microorganisms, which comprises a culture solution, adsorption resin, a culture bottle body, a bottle cap and a receptor;

the culture solution comprises tryptone, gelatin peptone, brain heart extract, L-arginine, L-cysteine hydrochloride, activity synergistic regulator, sodium citrate, potassium dihydrogen phosphate and vitamin C.

Further, the culture solution comprises: 1-10g/L tryptone, 1-10g/L gelatin peptone, 1-10g/L brain heart extract, 1-10g/L L-arginine, 1-10g/L L-cysteine hydrochloride, 0.08-0.3g/L activity synergistic regulator, 0.2-0.5g/L sodium citrate, 0.2-0.5g/L potassium dihydrogen phosphate and 0.8-2g/L vitamin C.

Preferably, the culture solution comprises: 5g/L tryptone, 5g/L gelatin peptone, 5g/L brain heart extract, 3.8g/L L-arginine, 2.4g/L L-cysteine hydrochloride, 0.2g/L activity synergistic regulator, 0.3g/L sodium citrate, 0.3g/L potassium dihydrogen phosphate and 1.2g/L vitamin C.

Further, the activity potentiator modulator includes phytosterols and hemin. It is to be noted that the phytosterols used in the present invention are all water-soluble phytosterols.

Preferably, the weight ratio of the phytosterol to the hemin is 1-2:3-5, and more preferably 1: 4.

Further, the phytosterol comprises one or more of sitosterol, stigmasterol and campesterol.

Furthermore, the adsorption resin is anion exchange resin and macroporous adsorption resin, and the anion exchange resin and the macroporous adsorption resin can be matched in any proportion for use.

Further, the sensor is positioned at the bottom of the culture bottle body and contains fluorescent dye.

Further, the culture solution was prepared using double distilled water.

The invention also provides application of the kit in-vitro detection of anaerobic microorganisms for non-diagnostic purposes.

Further, the anaerobic microorganisms include Clostridium histolyticum, Bacteroides fragilis, Bacteroides vulgatus, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, and Clostridium perfringens.

The technical effects obtained by the invention are as follows:

1. the liquid culture medium of the product of the invention is added with various nutrient components for the growth culture of various microorganisms, and the raw materials are matched with each other to achieve excellent effect. The culture bottle is filled with sterile gas to provide growth conditions for the microorganisms, so that the growth requirements of the microorganisms with different nutritional requirements are met. The resin is added to neutralize antibiotics and reduce the influence of the antibiotics on blood culture results.

2. The bacteria can release products such as carbon dioxide in the metabolic process, the carbon dioxide reacts with a receptor containing fluorescent dye at the bottom of the culture bottle, so that fluorescent substances combined with the carbon dioxide in the receptor are excited to emit fluorescence, a detection system of the full-automatic blood culture instrument automatically detects the fluorescence level every 10min, a culture result is obtained through processing of a computer data system, and the signals such as sound, light and the like are reported.

Detailed Description

It should be noted that the raw materials used in the present invention are all common commercial products, and thus the sources thereof are not particularly limited.

Example 1

An in vitro detection kit for anaerobic microorganisms, which is characterized in that: comprises a culture solution, adsorption resin, a culture bottle body, a bottle cap and a receptor;

wherein the culture solution comprises 1g/L tryptone, 1g/L gelatin peptone, 1g/L brain heart extract, 1g/L L-arginine, 1g/L L-cysteine hydrochloride, 0.08g/L activity synergistic regulator, 0.2g/L sodium citrate, 0.2g/L potassium dihydrogen phosphate and 0.8g/L vitamin C. The activity synergistic regulator comprises sitosterol and hemin in a weight ratio of 1: 3. The culture solution was prepared using double distilled water.

In addition, the adsorption resin is a mixture of anion exchange resin and macroporous adsorption resin with the volume ratio of 1:1, and the sensor is positioned at the bottom of the flask body of the culture flask and contains fluorescent dye.

Example 2

An in vitro detection kit for anaerobic microorganisms, which is characterized in that: comprises a culture solution, adsorption resin, a culture bottle body, a bottle cap and a receptor;

wherein the culture solution comprises 10g/L tryptone, 10g/L gelatin peptone, 10g/L brain heart extract, 10g/L L-arginine, 10g/L L-cysteine hydrochloride, 0.3g/L activity synergistic regulator, 0.5g/L sodium citrate, 0.5g/L potassium dihydrogen phosphate and 2g/L vitamin C. The active synergistic regulator comprises stigmasterol and hemin in a weight ratio of 2: 5. The culture solution was prepared using double distilled water.

In addition, the adsorption resin is a mixture of anion exchange resin and macroporous adsorption resin with the volume ratio of 1:1, and the sensor is positioned at the bottom of the flask body of the culture flask and contains fluorescent dye.

Example 3

An in vitro detection kit for anaerobic microorganisms, which is characterized in that: comprises a culture solution, adsorption resin, a culture bottle body, a bottle cap and a receptor;

wherein the culture solution comprises 5g/L tryptone, 5g/L gelatin peptone, 5g/L brain heart extract, 3.8g/L L-arginine, 2.4g/L L-cysteine hydrochloride, 0.2g/L activity synergistic regulator, 0.3g/L sodium citrate, 0.3g/L potassium dihydrogen phosphate and 1.2g/L vitamin C. The active synergistic regulator comprises campesterol and hemin in a weight ratio of 1: 4. The culture solution was prepared using double distilled water.

In addition, the adsorption resin is a mixture of anion exchange resin and macroporous adsorption resin with the volume ratio of 1:1, and the sensor is positioned at the bottom of the flask body of the culture flask and contains fluorescent dye.

Comparative example 1

The only difference from example 1 is that the culture broth comprises: 0.8g/L tryptone, 12g/L gelatin peptone, 0.8g/L brain heart extract, 12g/L L-arginine, 0.8g/L L-cysteine hydrochloride, 0.4g/L activity synergistic regulator, 0.1g/L sodium citrate, 0.6g/L potassium dihydrogen phosphate and 0.5g/L vitamin C.

Comparative example 2

The only difference from example 1 is that no activity enhancer was added.

Comparative example 3

The only difference from example 1 is that the weight ratio of phytosterol to hemin is 3:2 (the total weight of both is identical to example 1).

Test 1: universality of the kit of the invention

Sample requirements:

1. arterial blood or other sterile body fluid samples collected using the correct medical technique.

2. Special care must be taken in the sterile handling of the sample during collection.

3. The blood culture bottles should be sent to the laboratory within 2 hours. It is not recommended to leave the bottle at room temperature for several hours, and the blood culture bottle should not be refrigerated or frozen after inoculation; because this may kill some microorganisms and may also break up frozen liquid-filled bottles, blood culture specimens collected by lysis-centrifugation should not be left for more than 8 hours; too long a specimen placement time may reduce the positive rate or delay the growth of certain bacteria.

The detection method comprises the following steps:

1. and (5) checking whether the culture bottle is polluted or damaged.

2. The transparent plastic cover is opened, and the rubber cover is sterilized by alcohol cotton ball or iodine tincture.

3. Strictly performing aseptic operation, obtaining patient samples (5-10 mL of blood is recommended for adults and 1-3mL of blood is recommended for infants), inoculating the patient samples into a culture bottle under aseptic conditions, conveying the culture bottle to a microbiological chamber, inclining the culture bottle for multiple times, and immediately putting the culture bottle into a full-automatic detection instrument or an incubator for culture.

The product performance index is as follows:

1. appearance: the culture bottle has no crack, no leakage and no seepage; the liquid in the bottle has no turbidity, no flocculent precipitate, and is clear and transparent.

2. And (3) sterility: 5 bottles of culture bottles are taken and placed at the temperature of 35-37 ℃ for 7 days for aseptic growth.

3. Growth test:

the culture bottles in the examples 1-3 are inoculated with the quality control strains (Clostridium histolyticum, Bacteroides fragilis, Bacteroides vulgatus, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus and Clostridium perfringens) with the suspension concentration of 100cfu/mL-1000cfu/mL, and cultured for 24h-72h at 35-37 ℃.

Note that:

1. the flask is used for in vitro diagnostics only.

2. The culture bottle is a disposable product, and the used culture bottle can not be recycled.

3. The blood anticoagulant is already added into the culture bottle without adding any other blood anticoagulant.

4. In venipuncture and flask inoculation, care is taken to prevent contamination of the patient sample.

5. The inoculated blood culture bottle, the sample collection needle and the blood drawing instrument are considered to have infectious materials and must be processed according to the local laboratory procedures and specifications.

6. The culture bottle should be used within the expiration date indicated by the outer package.

And judging the condition by the result:

manual visual interpretation results: after being inclined, the culture bottle is vertically cultured for 24 to 72 hours in an incubator at the temperature of between 35 and 37 ℃, if the following phenomena occur: the culture solution is hemolyzed, becomes dark in color or has bacterial colonies growing out in a solid phase, and the like, which is positive, can be further used for strain identification and drug sensitivity experiments. Observing every day, if the phenomenon does not appear, continuing culturing, and if the phenomenon does not appear in one week, performing microscopic examination and blind transmission confirmation, and reporting a negative result after the sterility is confirmed.

Detecting and interpreting results by an instrument: the culture bottle is placed in the instrument after being scanned, if the instrument for culturing positive bacteria can automatically give an alarm, the culture bottle is positive, and strain identification and drug sensitivity test can be further carried out. Within 5 days, the instrument does not give an alarm, and negative result reports are made after microscopic examination and blind transmission confirm that the instrument is sterile.

TABLE 1

As is clear from Table 1, after the culture by the kit of examples 1 to 3 of the present invention, the results of the artificial visual observation and the results of the instrumental detection and interpretation of each species were all the growth of the microorganism, and the positive was reported. Therefore, the kit disclosed by the invention has effectiveness and universality for detecting each strain.

Test 2: accuracy of the kit of the invention

Three anaerobic bacteria (clostridium histolyticum, bacteroides fragilis and bacteroides vulgatus) are randomly selected, the kit in different examples is adopted for detection, 20 groups of each test are carried out, the positive detection rate (the bacterial suspension concentration is 100cfu/mL) is counted, and the average detection time for detecting the positive bacteria is calculated to obtain tables 2 and 3.

TABLE 2

TABLE 3

As can be seen from tables 2 and 3, the kit of the present invention can effectively shorten the detection time of Clostridium histolyticum, Bacteroides fragilis and Bacteroides vulgatus, and increase the positive detection rate, wherein the detection time of the kit of examples 1-3 for each strain is between 9-14h, and the positive detection rate is up to 100%. In comparison, the proportion of each group does not reach a proper state due to the selection of the raw material components in the culture solution and the matching condition, the final detection time is long, and the positive detection rate does not reach a permitted value.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.