WO2022141942A1 - Bacillus cereus standard strains containing specific molecular target, and detection and use thereof - Google Patents

Abstract

The present invention relates to the technical field of bioengineering, and in particular to five standard strains of Bacillus cereus which appear in China Autonomous Intellectual Property and conform to Domestic Propagation Law. In addition, same can be used as the standard reference strains in different fields such as food, medicine, clinical examination and the like. The deposit numbers thereof are GDMCC 60865, GDMCC 60866, GDMCC 60867, GDMCC 60868 and GDMCC 60869, respectively. Five standard strains have typical physiological and biochemical characteristics, have clear information in the sample source, the genetic background, the drug resistance, the virulence gene and the like, and completely meet the requirements of food, medicine and clinical examination on a standard strain. The present invention also relates to a set of specific target genes for detecting and identifying the above-mentioned five standard strains, and the corresponding PCR primers. Finally, also provided in the present invention is a freeze-drying protective agent for Bacillus cereus with a high survival rate and specificity, which can be used for long-term storage of the standard strains of the present invention.

Description

Bacillus cereus standard strain containing specific molecular target and its detection and application technical field

The invention belongs to the technical field of bioengineering, and specifically relates to five Bacillus cereus standard reference strains that can be used in different fields such as food, medicine, and clinical testing, and molecular target genes for detecting and identifying the five Bacillus cereus standard strains and PCR amplification primers. In addition, a novel freeze-drying protection agent which is convenient for long-term storage of standard strains of Bacillus cereus is provided.

Background technique

Bacillus cereus is a common food-borne pathogen in my country. According to the "2008-2015 my country Food Poisoning Bulletin" published by the National Health and Family Planning Commission, food poisoning events caused by Bacillus cereus ranked third in food poisoning events caused by food-borne pathogens. , is dangerous. Bacillus cereus mainly contaminates foods with high starch content, such as rice, rice noodles, etc., which can cause two different types of food poisoning: diarrhea-type and vomiting-type, which are caused by the diarrhea toxin (enterotoxin) and vomitoxin it produces respectively. In addition, Bacillus cereus is also the number one threat to the dairy industry, often causing contamination of the dairy production chain and final products, not only causing serious economic losses, but also bringing great potential safety hazards. In my country, food poisoning caused by Bacillus cereus is mainly caused by DON (about 75%), and even severe cases can lead to death. Due to its wide distribution and the ability to produce resistant spores, Bacillus cereus has not only become the most common contamination and pathogenic risk in the food industry chain, but also one of the common risks in pharmaceuticals and clinical practice. Therefore, Bacillus cereus should be the key monitoring target for foodborne pathogens. Establishing a suitable detection, monitoring and control system is of great significance to food, medicine and clinical aspects. The use of standard strains and the rationality of standard strain selection determine the reliability and implementation of the relevant systems. Standard strains are preserved by international or domestic culture collection centers, and the biological characteristics are confirmed and guaranteed and can be traced back.

At present, most of the standard strains of Bacillus cereus used in the actual supervision and monitoring process are strains from the American Collection of Microbial Strains. Due to the differences in eating habits in Europe and America, food poisoning events caused by Bacillus cereus are few, and are not regarded as key monitoring objects. Therefore, few strains are isolated and collected, which cannot well reflect the transmission characteristics of this bacteria in food. Chinese residents take rice, rice noodles, etc. as their staple food. The detection rate of Bacillus cereus has been at a high level, but there is a lack of representative isolates as standard strains with independent intellectual property rights to study the genetic characteristics of this bacteria in China. and propagation laws.

SUMMARY OF THE INVENTION

[Corrected 12.06.2021 according to Rule 26]
In order to solve the above technical problems, the present invention provides 5 standard strains of Bacillus cereus isolated from different food types in China. The strain has typical physiological and biochemical characteristics of Bacillus cereus, and can better reflect the genetic background of the bacteria in China, and can make up for the vacancy of food-borne Bacillus cereus standard strains without local isolation sources in China. Strain 2801 is isolated from pork samples, its preservation number is: GDMCC 60865, and its classification name is: Bacillus cereus; 260-1B is isolated from a cold noodle sample, and its preservation number is: GDMCC 60867, and its classification name is: Bacillus cereus; strain- 1761-2A was isolated from the fried rice sample, and its preservation number is: GDMCC 60868, and its classification name is: Bacillus cereus; strain-Y1712 is isolated from a fried rice sample, and its preservation number is: GDMCC 60869, and its classification name is: Bacillus cereus; 2841-1B was isolated from the cucumber sample, its deposit number is: GDMCC 60866, and the classification name is: Bacillus cereus. The 5 strains of Bacillus cereus isolated above are all preserved in the Guangdong Provincial Microbial Culture Collection Center, Address: Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China, zip code: 510075, preservation date: October 2019 27th.

The technical scheme adopted in the present invention is:

The present invention provides specific molecular targets for detecting Bacillus cereus standard strains, the molecular targets are:

(a) corresponding one or more nucleotide sequences shown in SEQ ID NOs: 1 to 10; or,

(b) The nucleotide sequence in (a) is substituted, deleted or added by one or several nucleotides and has more than 90% homology with the nucleotide in (a). The newly obtained Bacillus cereus in the present invention obtains specific molecular targets contained in the present invention through pan-genome analysis and comparison, and can specifically detect the standard strains of Bacillus cereus contained in the present invention, with strong specificity.

The present invention provides primers for detecting the specific molecular target as claimed in claim 1, and the PCR amplification primers for the nucleotide sequence as shown in SEQ ID NO.1 include: upstream as shown in SEQ ID NO.11 Primers and downstream primers as shown in SEQ ID NO.12; PCR amplification primers for the nucleotide sequence shown in SEQ ID NO.2 include: upstream primers as shown in SEQ ID NO.13 and as shown in SEQ ID The downstream primer shown in NO.14; the PCR amplification primer for the nucleotide sequence shown in SEQ ID NO.3 includes the upstream primer shown in SEQ ID NO.15 and the upstream primer shown in SEQ ID NO.16 Downstream primers; PCR amplification primers for the nucleotide sequence shown in SEQ ID NO.4 include upstream primers as shown in SEQ ID NO.17 and downstream primers as shown in SEQ ID NO.18; for SEQ ID NO.18 The PCR amplification primers of the nucleotide sequence shown in ID NO.5 include the upstream primer shown in SEQ ID NO.19 and the downstream primer shown in SEQ ID NO.20; for the nucleotide sequence shown in SEQ ID NO.6 The PCR amplification primers of the nucleotide sequence include the upstream primer shown in SEQ ID NO.21 and the downstream primer shown in SEQ ID NO.22; for the nucleotide sequence shown in SEQ ID NO.7 PCR amplification primers include upstream primers as shown in SEQ ID NO.23 and downstream primers as shown in SEQ ID NO.24; PCR amplification primers for the nucleotide sequence shown in SEQ ID NO.8 include as shown in The upstream primer shown in SEQ ID NO.25 and the downstream primer shown in SEQ ID NO.26; the PCR amplification primers for the nucleotide sequence shown in SEQ ID NO.9 include those shown in SEQ ID NO.27. The upstream primer shown in SEQ ID NO.28 and the downstream primer shown in SEQ ID NO.28; the PCR amplification primers for the nucleotide sequence shown in SEQ ID NO.10 include the upstream primer shown in SEQ ID NO.29 and the upstream primer shown in SEQ ID NO.10. Downstream primer shown in SEQ ID NO.30.

The present invention also provides a group of Bacillus cereus standard strains, which are (a), (b), (c), (d) or (e):

(a) strain 2801, containing at least one nucleotide sequence as shown in SEQ ID NOs: 1 to 3;

(b) strain 260-1B, containing the nucleotide sequence as shown in SEQ ID NO:4;

(c) strain 1761-2A, containing the nucleotide sequence shown in SEQ ID NO:5;

(d) strain Y1712, containing at least one nucleotide sequence shown in SEQ ID NOs: 6-8;

(e) Strain 2841-1B, containing at least one nucleotide sequence as shown in SEQ ID NOs: 9-10.

Preferably, the strain-2801 carries the following virulence genes: nheA, nheB and nheC; antibiotics resistant to it include: ampicillin, cefotaxime, cefoxitin, penicillin, amoxicillin-clavulanate, Rifampicin, dalfopristin, and nitrofurantoin.

Preferably, the strain-260-1B carries the following virulence genes: hblA, hblC, hblD, nheA, nheB, nheC and cytK; the antibiotics it tolerates include: ampicillin, amoxicillin-clavulanate , penicillin, cefotaxime, cefoxitin, co-trimoxazole, rifampicin, dalfopristin, and nitrofurantoin.

Preferably, the strain-1761-2A carries the following virulence genes: nheA, nheB and nheC; antibiotics resistant to it include: ampicillin, amoxicillin-clavulanate, penicillin, cefotaxime, cefoxicil tetracycline, tetracycline, and rifampicin.

Preferably, the strain-Y1712 carries the following virulence genes: nheA and cytK; the antibiotics it tolerates include: ampicillin, amoxicillin-clavulanate, penicillin, cefotaxime, cefoxitin, tetracycline and rifampicin.

Preferably, the strain-2841-1B carries the following virulence genes: hblA, hblC, hblD, nheA, nheB, nheC, cytK and cesB; the antibiotics it tolerates include: ampicillin, cefotaxime, cefoxime Ding, penicillin and rifampicin.

Preferably, the deposit number of the strain-2801 is GDMCC 60865; the deposit number of the strain-260-1B is: GDMCC 60867; the deposit number of the strain-1761-2A is GDMCC 60868; The deposit number is GDMCC 60869; the deposit number of the strain-2841-1B is GDMCC 60866.

The invention also provides the application of the standard strain in the study of antibiotic resistance of Bacillus cereus. Preferably, the antibiotic resistance of the strain-2801 is resistance to ampicillin, cefotaxime, cefoxitin, penicillin, amoxicillin-clavulanate, rifampicin, dalfopristin and nitrofurantoin; The antibiotic resistance of the described strain-260-1B is resistance to ampicillin, amoxicillin-clavulanate, penicillin, cefotaxime, cefoxitin, co-trimoxazole, rifampicin, dalfopristin and nitrofurantoin The strain-1761-2A antibiotic resistance is resistance to ampicillin, amoxicillin-clavulanate, penicillin, cefotaxime, cefoxitin, tetracycline and rifampicin; the strain-Y1712 Antibiotic resistance was resistance to ampicillin, amoxicillin-clavulanate, penicillin, cefotaxime, cefoxitin, tetracycline, and rifampicin; the strain-2841-1B antibiotic resistance was ampicillin Resistance to cillin, cefotaxime, cefoxitin, penicillin and rifampicin.

The invention also provides the application of the Bacillus cereus in improving the accuracy of testing the Bacillus cereus color plate.

The invention also provides a freeze-drying protective agent for Bacillus cereus, which is characterized by comprising the following components in parts by weight: 0.1-10 parts of cellobiose, 5-10 parts of skim milk powder, and 0.1-3 parts of polyvinylpyrrolidone part, sodium glycerophosphate 0.3-1 part, phytate 0.1-2 part.

The reason why the above components are selected in the present invention is that cellobiose and sodium glycerophosphate have a polyhydroxy structure, which can form hydrogen bonds with the polar groups of bacterial proteins, replace water molecules, and act as dehydration protection agents in the process of drying and dehydration. Function; skimmed milk powder can be wrapped in the outer layer of bacteria cells to protect the bacteria; polyvinylpyrrolidone can not only reduce the freezing and dehydration denaturation caused by the ice-water interfacial tension during the freezing and dehydration process, but also reduce the active components during the rehydration process. Acts as a wetting agent and a refolding agent; phytate is an antioxidant that can protect the activity of cellular oxidase during freeze-drying and storage, preventing oxidative deterioration of freeze-dried products.

Preferably, the freeze-drying protective agent comprises the following components in parts by weight: 7 parts of cellobiose, 0.5 parts of sodium glycerophosphate, 5 parts of skim milk powder, 2 parts of polyvinylpyrrolidone, and 1 part of phytic acid.

The beneficial effects of the present invention are as follows: the standard strain of Bacillus cereus created in the present invention has more detailed physiological and biochemical index information, including strain isolation source, sampling date and location, biochemical identification characteristics, Bacillus cereus The carrying status of virulence genes, antibiotic resistance characteristics, MLST typing characteristics, etc., are convenient for scientific research, production and other fields to be used as reference and comparison in different research directions; the present invention screened out specific molecular target genes for established standard strains , and with detailed PCR amplification primers and systems, it is convenient to quickly and accurately identify the standard strain.

In addition, for the Bacillus cereus of the present invention, the inventor provides a freeze-drying protection agent. The protective agent has the following advantages: good shape, beautiful appearance and good water solubility, can be completely dissolved within 1-2 seconds; freeze-dried survival rate can reach more than 40%; The order of magnitude of viable bacteria does not change for at least one year and can be used for long-term storage of quality control strains.

Description of drawings

Fig. 1 is a schematic diagram of the specific detection results of 3 target genes of standard strain 2801 (where a is the result of specific amplification detection of different Bacillus cereus environmental isolates with primers designed by the specific target gene 2801_03520 of standard strain 2801 b with the primers designed by the specific target gene 2801_03520 of the standard strain 2801 to carry out specific amplification detection to other strains of different bacterial genera; c with the primers designed by the specific target gene 2801_03513 of the standard strain 2801 to different Bacillus cereus The results of specific amplification and detection of environmental isolates; d, the results of specific amplification and detection of other strains of different genus with primers designed by the specific target gene 2801_03513 of standard strain 2801; e using the specific target gene of standard strain 2801 The results of specific amplification and detection of different Bacillus cereus environmental isolates with primers designed by 2801_02920; f using primers designed by the specific target gene 2801_02920 of standard strain 2801 to specifically amplify and detect other strains of different genus result).

Fig. 2 is the specific detection result of the specific target gene of the standard strain 260-1B (wherein a uses the primers designed by the specific target gene 260-1B_04473 of the standard strain 260-1B to carry out specific amplification of different Bacillus cereus environmental isolates The results of amplification detection; b the results of specific amplification detection of other strains of different strains with primers designed for the specific target gene 260-1B_04473 of the standard strain 260-1B).

Fig. 3 is the specific detection result of the target gene of standard strain 1761-2A (wherein a uses primers designed for the specific target gene 1761-2A_03332 of standard strain 1761-2A to carry out specific amplification detection on different Bacillus cereus environmental isolates Result; b The results of specific amplification detection of other strains of different strains with primers designed for the specific target gene 1761-2A_03332 of the standard strain 1761-2A).

Fig. 4 is the specificity detection result of 3 target genes of standard strain Y1712 (wherein a uses the primers designed by the specific target gene Y1712_05212 of standard strain Y1712 to carry out specific amplification detection to different Bacillus cereus environmental isolates; b The results of specific amplification and detection of other strains of different genus with the primers designed by the specific target gene Y1712_05212 of the standard strain Y1712; The results of specific amplification and detection of the isolated strain; d, the results of specific amplification and detection of other strains of different genus with primers designed by the specific target gene Y1712_01910 of the standard strain Y1712; e, the specific target gene Y1712_02045 of the standard strain Y1712 The results of specific amplification and detection of different P. cereus environmental isolates with the designed primers; f The results of specific amplification and detection of other strains of different genus with primers designed with the specific target gene Y1712_02045 of the standard strain Y1712 )

Figure 5 is the specific detection results of the two target genes of the standard strain 2841-1B (where a primers designed by the specific target gene 2841-1B_00711 of the standard strain 2841-1B are used to carry out specificity for different Bacillus cereus environmental isolates The results of amplification detection; b the results of specific amplification detection of other strains of different strains with primers designed by the specific target gene 2841-1B_00711 of the standard strain 2841-1B; c using the specific target gene of the standard strain 2841-1B The results of specific amplification and detection of different Bacillus cereus environmental isolates with primers designed by 2841-1B_00745; d. The primers designed by the specific target gene 2841-1B_00745 of the standard strain 2841-1B were used to perform specific amplification tests on other strains of different bacteria. results of specific amplification assays).

Figure 6 is a schematic diagram of the changes in the storage period of Bacillus cereus quantitative quality control bacteria.

Detailed ways

In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments and accompanying drawings.

Example 1 Isolation and culture of standard strains of Bacillus cereus

The collected food samples were thoroughly cut into pieces under aseptic conditions, and 25 g of the samples were weighed and placed in a sterile homogenizing cup filled with 225 mL of PBS buffer or physiological saline. min～10000r/min homogenize for 1min～2min, shake and mix evenly, as a 1:10 sample homogenate solution. Aspirate 1 mL of 1:10 sample homogenate solution, add it to a dilution tube containing 9 mL of PBS or normal saline, and mix thoroughly to make 1:100 sample homogenate solution. Select the sample homogenate (including the original solution) of the above three dilutions, and transfer them to three MYP agar plates with 0.3mL, 0.3mL and 0.4mL inoculum respectively, then coat the whole plate with a sterile L rod and let it stand for 10min. If the sample solution is not easily absorbed, the plate can be placed in an incubator at 30°C±1°C for 1 hour. After the sample is evenly absorbed, the plate can be turned over, placed in the incubator, and incubated at 30°C±1°C for 24h±2h. If the colony is not typical, continue to cultivate for 24h±2h and observe again. On MYP agar plates, typical colonies are pinkish (indicating non-fermentation of mannitol) surrounded by a white to pale pink precipitation ring (indicating lecithinase production). Pick at least 5 typical colonies (less than 5) from each plate, streak them on nutrient agar plates for pure culture, and culture at 30℃±1℃ for 24h±2h. Typical colonies were inoculated into tryptone soybean broth medium, cultured at 30°C and 200 rpm for 12 hours, and the bacterial solution was added to a 25% glycerol tube under aseptic conditions, and stored in a -80°C refrigerator. The purified colonies can be used for subsequent screening experiments as well as biochemical identification and molecular typing.

The sample sources and sampling location information of the 5 strains of Bacillus cereus obtained above are shown in Table 1 below:

Table 1 Sample source and sampling location information of 5 strains of Bacillus cereus

strain name	deposit number	Sampling location	Sample source
Strain 2801	GDMCC 60865	Changsha City, Hunan Province, China	pork
Strain 260-1B	GDMCC 60867	Shenzhen City, Guangdong Province, China	cold noodles
Strain 1761-2A	GDMCC 60868	Zhanjiang, Guangdong, China	fried noodles
Strain Y1712	GDMCC 60869	Shantou, Guangdong, China	fried rice
Strain 2841-1B	GDMCC 60866	Changsha City, Hunan Province, China	cucumber

Example 2 Characteristic sequence analysis of standard strains of Bacillus cereus

The 5 strains of Bacillus cereus obtained in Example 1 were subjected to specific sequence analysis, and the specific implementation steps were as follows:

(1) Genome extraction

Collect bacteria by centrifugation, add lysozyme and glass beads simultaneously to remove cell walls, add protease and lysate to lyse bacteria, add ethanol and mix evenly to adsorb DNA on the column, wash to remove impurities, and add eluent to elute DNA. After eluting the DNA, place it in a -40°C refrigerator for later use.

(2) Whole genome sequencing

The DNA samples of the extracted standard strains were used for whole genome sequencing using the Thermo Fisher Scientific Ion S5 genome sequencing platform. The sequence files obtained by sequencing were assembled using Spades software, and the genome sequences obtained after assembly were annotated using Prokka software.

(3) Pan-genome analysis of specific molecular target genes for standard strains

Roary software was used to perform pan-genome analysis on the whole genome sequence annotation results of standard strains and other 155 Bacillus cereus strains. The script extracts the base sequence of the target gene.

(4) PCR primer design for specific molecular target genes of standard strains

According to the base sequences of each specific molecular target gene of the 5 standard strains obtained by the pan-genome analysis, the Primer-BLAST primer design tool on the NCBI official website was used to design the corresponding PCR amplification primers to be specific for different target genes respectively. Sexual Amplification Detection. The target genes of 5 standard strains in the present invention and their amplification primer information are shown in Table 2:

Table 2 Standard strain-specific molecular target gene sequences and their PCR primer information

(5) PCR detection of standard strains and other strains of the same genus and different genus is performed using the synthesized primers to verify the specificity of the screened molecular target genes and primers. The information of PCR reaction system and reaction program is shown in Tables 3 and 4 below:

Table 3 PCR amplification system of standard strain-specific molecular target genes

Table 4 PCR reaction procedures of standard strain-specific molecular target genes

Note: The extension time in the table varies with the amplified gene, and is converted with reference to the length of different target genes in Table 2.

The strain information of different bacterial genera selected for PCR verification is shown in Table 5 and Table 6:

Table 5 The strain information of different genera selected for the specific identification and detection of standard strains

Numbering	strain name		Genus
1	ATCC29544		Enterobacter sakazakii
2	CMCC54002		Listeria monocytogenes
3	CMCC54004		Listeria monocytogenes
4	CMCC54007		Listeria monocytogenes
5	ATCC19115		Listeria monocytogenes
6	CMCC51592	Shigella sonnei
7	ATCC25922		Escherichia coli
8	ATCC9027		Pseudomonas aeruginosa
9	ATCC10104		Pseudomonas aeruginosa
10	ATCC15442		Pseudomonas aeruginosa
11	ATCC27853		Pseudomonas aeruginosa
12	ATCC25923		Staphylococcus aureus
13	ATCC29213		Staphylococcus aureus
14	ATCC14028		Salmonella typhimurium
15	ATCC50335		Salmonella Enteritidis
16	23201201	Vibrio parahaemolyticus
17	23201233	Vibrio parahaemolyticus
18	ATCC10206	Bacillus mycoides

19	ATCC6633	Bacillus subtilis
20	ATCC10792	Bacillus thuringiensis
twenty one	ATCC14579	Bacillus cereus
twenty two	CMCC63303	Bacillus cereus

Table 6 Information on Bacillus cereus isolates selected for specific identification and detection of standard strains

Numbering	strain name		Genus
1	137-Bc		Bacillus cereus
2	160-1-Bc		Bacillus cereus
3	184-2-Bc		Bacillus cereus
4	Y343-Bc		Bacillus cereus
5	561-3B-Bc		Bacillus cereus
6	Y562-Bc		Bacillus cereus
7	1128-Bc		Bacillus cereus
8	1247-Bc		Bacillus cereus
9	1360-Bc		Bacillus cereus
10	Y1589-1-Bc		Bacillus cereus
11	1610-1A-Bc		Bacillus cereus
12	1634-1A-Bc		Bacillus cereus
13	1634-3A-2-Bc		Bacillus cereus
14	1659-3B-Bc		Bacillus cereus
15	1686-1C-Bc		Bacillus cereus
16	Y1688-Bc		Bacillus cereus
17	1712-1A-Bc		Bacillus cereus
18	2039-1-Bc		Bacillus cereus
19	2818-2C-Bc		Bacillus cereus
20	2829-1A-Bc	Bacillus cereus
twenty one	3560-1A-Bc	Bacillus cereus
twenty two	3715-Bc	Bacillus cereus

The PCR detection results for each specific molecular target gene of the five standard strains are shown in Figures 1-5. From the detection results, using the PCR primers for each specific molecular target gene of the five standard strains, only specific bands with the same fragment length were amplified from the genomic DNA of the corresponding standard strains, indicating that these primers have good specificity , no cross-amplification reaction occurs, and can be used as primers for the specific identification and detection of 5 standard strains of Bacillus cereus.

(6) The products obtained by PCR amplification of standard strains were sent to Huada Gene Company for sequencing to determine the validity of primer amplification results. The sequencing results showed that they were consistent with the specific molecular target gene sequences screened in the previous stage.

Example 3 Physiological and biochemical characteristics analysis of Bacillus cereus standard strains

1. Biochemical identification results of standard strains

Biochemical identification was carried out according to GB 4789.14-2014 "National Food Safety Standard for Food Microbiological Inspection of Bacillus cereus", and a single colony of pure culture was picked for catalase test, kinetic test, nitrate reduction test, and lysozyme resistance. Test, V-P test, glucose utilization (anaerobic) test, root growth test, hemolysis test, protein toxin crystallization test, yolk reaction test, casein decomposition test. The standard strain of Bacillus cereus (ATCC14579), the standard strain of Bacillus mycoides (ATCC10206) and the standard strain of Bacillus thuringiensis (ATCC10792) were used as controls to conduct simultaneous experiments, and the biochemical identification results are as follows (Table 7)

Table 7 Record table of biochemical identification results of standard strains

Note: "+" indicates 90%-100% positive strains; "-" indicates 90%-100% negative strains. "+/-" means that most strains were positive; "-/+" means that most strains were negative; "/" means that no test was performed.

Example 4 Virulence gene carrying characteristics of 5 standard strains of Bacillus cereus

The virulence gene carrying status of Bacillus cereus standard strains was confirmed by PCR. Seven enterotoxin genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK) related to diarrhea symptoms caused by Bacillus cereus and DON synthase gene (cesB) related to vomiting symptoms were selected for detection. The PCR amplification detection system, reaction conditions and primer information are shown in Tables 8 to 10 below. The primers were synthesized by Shanghai Sangon Biological Co., Ltd. The virulence gene detection results of the five standard strains are shown in Table 11 below.

Table 8 PCR amplification system of standard strain-specific molecular target genes

System components	volume
Strain DNA Template	1μL
PCR Mix	12.5μL
forward primer	1μL
reverse primer	1μL
ddH2O	9.5μL
total capacity	25μL

Table 9 PCR reaction procedures for standard strain-specific molecular target genes

Note: The annealing temperature in the table varies with the amplified genes and primers, see Table 10 for details.

Table 10 Primers used in the detection of Bacillus cereus virulence genes

Table 11 The virulence gene carrying situation of 5 standard strains of Bacillus cereus

Standard strain name	virulence gene carrier
2801	nheA-nheB-nheC
260-1B	hblA-hblC-hblD-nheA-nheB-nheC-cytK
1761-2A	nheA-nheB-nheC
Y1712	nheA-cytK
2841-1B	hblA-hblC-hblD-nheA-nheB-nheC-cytK-cesB

Example 5 Drug susceptibility characteristics of Bacillus cereus standard strains

Referring to the American Clinical and Laboratory Standards Institute (CLSI) standard for the detection of Staphylococcus aureus, the antibiotic susceptibility of Bacillus cereus standard strains was evaluated by KB disc diffusion method. The antibiotic susceptibility paper was pasted on the MH agar plate coated with the bacterial solution. The drug in the paper diffused in the agar to form a concentration gradient, while the strains within the inhibitory concentration range around the paper could not grow, and the bacteria outside the inhibitory range could not grow. The strains can grow to form a transparent antibacterial zone around the paper, and the size of the antibacterial zone can reflect the sensitivity of the test bacteria to the drug. See Table 12 for specific information on antibiotic susceptibility discs.

Specific steps are as follows:

1) Activation of the bacteria to be tested. It was inoculated into tryptone soybean broth, cultured for 24 hours, streaked on a nutrient agar plate, and cultured overnight.

2) Prepare a suspension. Pick pure fresh colonies, resuspend in normal saline, and then use 0.5 McFarland turbidimetric tube to make the concentration equivalent.

3) Bacteria liquid coating. Pipette 1 mL of the suspension onto the MH agar plate and spread it evenly with a cotton swab. Then paste the susceptibility paper of different antibiotics according to the pre-marked area and make a mark. Placed in a constant temperature incubator at 37°C and incubated for 1 d.

4) Measurement of inhibition zone. Observe the experimental results and measure the size of the inhibition zone with a bacteriostatic zone measuring instrument or a ruler. Finally, referring to the American Clinical and Laboratory Standards Institute (CLSI) drug susceptibility test standards for Staphylococcus aureus (CLSI, 2010), the bacterial susceptibility, moderate resistance and resistance to antibiotics were judged. The antibiotic resistance test results of the 5 standard strains are shown in Table 13 below.

Table 12 Information on 20 antibiotics and evaluation criteria for the zone of inhibition

Table 13 Antibiotic resistance of 5 standard strains of Bacillus cereus

Standard strain	antibiotic resistance
2801	AMP-KF-FOX-P-AMC-RD-QD-FD
260-1B	AMP-AMC-P-KF-FOX-SXT-RD-QD-FD
1761-2A	AMP-AMC-P-KF-FOX-TE-RD
Y1712	AMP-AMC-P-KF-FOX-TE-RD
2841-1B	AMP-KF-FOX-P-RD

Example 6 Multi-locussequence typing analysis of Bacillus cereus standard strains

Primer sequence information for the seven housekeeping genes of Bacillus cereus was obtained from the PubMLST database (https://pubmlst.org/organisms/bacillus-cereus/primers) (Table 15). According to the primer information, the housekeeping gene sequence is amplified and sequenced, and the corresponding housekeeping gene sequence is obtained after splicing, and then uploaded to the MLST database for comparison, and the sequence code (allele number) of the gene is obtained. The sequences of each strain were arranged into allele profiles according to glpF, gmk, ilvD, pta, pur, pycA and tpi, and then the ST type of the strain was queried through the MLST database. The MLST-PCR amplification detection system, reaction conditions and primer information are shown in Tables 14-16 below. The primers were synthesized by Shanghai Sangon Biological Co., Ltd. The MLST detection results of the 5 standard strains are shown in Table 17 below.

Table 14 Standard strain MLST-PCR amplification system

System components	volume
Strain DNA Template	1μL
TakaRa Primer STAR Max	12.5μL
forward primer	1μL
reverse primer	1μL
ddH 2 O	9.5μL
total capacity	25μL

Table 15 Standard strain MLST-PCR reaction program

Note: The annealing temperature in the table varies with the amplified genes and primers, see Table 16 for details.

Table 16 Amplification primer information of 7 MLST housekeeping genes of Bacillus cereus

Table 17 MLST typing results of 5 standard strains of Bacillus cereus

Standard strain name		ST type
2801	ST1438
260-1B	ST1431
1761-2A	ST26
Y1712	ST90
2841-1B	ST1882

The present invention also provides a lyophilized protective agent of a Bacillus cereus standard strain, and the components of the lyophilized protective agent in different embodiments and comparative examples are shown in Table 18:

Table 18 Components of each group of lyoprotectants

In the comparative example, the weight part of skim milk powder was increased to make up the total amount due to its lack of components.

The freeze-dried survival rate of the freeze-dried bacterial species (GDMCC 60865) of the protective agents of Examples 7 to 9 and Comparative Examples 1 to 3 will be used, and the specific method is as follows:

After the strains are recovered, they are inserted into the culture medium and cultivated to the late logarithmic stage to the early stage of the stable stage, and an appropriate amount of bacteria is selected to be added to the protective agents of Examples 7-9 and Comparative Examples 1-3, and then dispensed into vials after mixing evenly. , and sample for dilution count, which is the bacterial content A0 before freeze-drying. Transfer the half-stoppered vial to the freeze dryer for pre-freezing, the pre-freezing temperature is -40 ° C, the time is 3 hours, the main drying is turned on, the time is 20-25 hours, and then the analysis and drying stage is entered, and the time is: After 6-8 hours, the drying is completed, the plug is pressed in a vacuum state and the freeze dryer is removed, and automatic capping is performed to ensure the complete vacuum state of the sample, and it is stored at -20°C. Take the sample after freeze-drying and carry out dilution count, the count result is the bacterial content A after freeze-drying, and the freeze-drying survival rate is the percentage of A and A0, and the results are shown in Table 19:

Table 19 Comparison of lyophilized survival rates of lyophilized protective agents with different compositions

As can be seen from Table 19, the lyophilized protective agent of Example 9 is the best embodiment of the present invention, therefore, taking Example 9 as a comparison object, the lyophilized protective agents of Comparative Examples 1 to 3 were prepared, and the results are as shown in Table 19 As shown, since Comparative Examples 1 to 3 lacked one of the components of the freeze-drying protective agent of the present invention, their protective effects were all worse than those of the examples, thus illustrating the component fibers in the freeze-drying protective agent of the present invention. There is a synergistic effect among disaccharide, polyvinylpyrrolidone and sodium glycerophosphate, and the effect of the protective agent of the present invention cannot be achieved without any one of them.

The freeze-drying stability will be compared with the freeze-dried strains of the protective agents in Examples 7-9 and Comparative Examples 1-3, and the specific methods are as follows:

According to the above preparation method, different protective agents were used to prepare quantitative quality control bacteria, and they were stored at -20°C, and 3 bottles were extracted every month to check the bacterial content according to the aforementioned counting method. In order to better compare the effect of each protective agent in the long-term storage process, when preparing quantitative quality control bacteria, the number of viable bacteria before freeze-drying was calculated according to the freeze-drying survival rate of each protective agent, so that after each protective agent was freeze-dried, The bacterial content is about 3000cfu/bottle. The changes in bacterial content of the quantitative quality control bacteria prepared by each protective agent after 12 months of storage are shown in Figure 6 below.

It can be seen from accompanying drawing 6 that after 12 months of storage, the bacterial content protected by the lyophilized protective agents of Examples 7 to 9 has no significant change, while the bacterial content protected by the lyophilized protective agents of Comparative Examples 1 to 3 has no significant change after 12 months of storage. The bacterial content decreased significantly after 2 months, and the bacterial content was close to 0 after 12 months.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. A specific molecular target for detecting a Bacillus cereus (Bacillus cereus) standard strain, wherein the molecular target is:

   (a) any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 10; or,

   (b) The nucleotide sequence in (a) is substituted, deleted or added by one or several nucleotides and has more than 90% homology with the nucleotide in (a).
2. The primer for detecting a specific molecular target as claimed in claim 1, wherein the PCR primer for amplification of the nucleotide sequence as shown in SEQ ID NO:1 comprises: upstream as shown in SEQ ID NO:11 Primers and downstream primers as shown in SEQ ID NO: 12; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO: 2 include: upstream primers as shown in SEQ ID NO: 13 and as shown in SEQ ID Downstream primers shown in NO: 14; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 3 include the upstream primers shown in SEQ ID NO: 15 and the upstream primers shown in SEQ ID NO. 16 Downstream primers; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:4 include upstream primers as shown in SEQ ID NO:17 and downstream primers as shown in SEQ ID NO:18; for SEQ ID NO:18 The PCR primers for amplification of the nucleotide sequence shown in ID NO: 5 include the upstream primer shown in SEQ ID NO: 19 and the downstream primer shown in SEQ ID NO: 20; The PCR primers for the amplification of the nucleotide sequence include the upstream primer as shown in SEQ ID NO:21 and the downstream primer as shown in SEQ ID NO.22; for the amplification of the nucleotide sequence as shown in SEQ ID NO:7 The increased PCR primers include upstream primers as shown in SEQ ID NO: 23 and downstream primers as shown in SEQ ID NO: 24; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO: 8 include as shown in SEQ ID NO: 8. The upstream primer set forth in SEQ ID NO:25 and the downstream primer set forth in SEQ ID NO:26; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:9 include those set forth in SEQ ID NO:27. The upstream primers shown in SEQ ID NO: 28 and the downstream primers shown in SEQ ID NO: 28; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 10 include the upstream primers shown in SEQ ID NO: 29 and as shown in SEQ ID NO: 10. Downstream primer shown in SEQ ID NO:30.
3. A standard strain of Bacillus cereus, characterized in that it is (a), (b), (c), (d) or (e):

   (a) strain 2801 contains at least one nucleotide sequence as shown in SEQ ID NOs: 1-3;

   (b) strain 260-1B, containing the nucleotide sequence as shown in SEQ ID NO:4;

   (c) strain 1761-2A, containing the nucleotide sequence shown in SEQ ID NO:5;

   (d) strain Y1712, containing at least one nucleotide sequence shown in SEQ ID NOs: 6-8;

   (e) Strain 2841-1B, containing at least one nucleotide sequence as shown in SEQ ID NOs: 9-10.
4. The standard strain of Bacillus cereus according to claim 3, wherein the strain 2801 carries the following virulence genes: nheA, nheB and nheC; antibiotics resistant to it include: ampicillin, cefotaxime, Cefoxitin, penicillin, amoxicillin-clavulanate, rifampicin, dalfopristin, and nitrofurantoin.
5. The standard strain of Bacillus cereus according to claim 3, wherein the strain 260-1B carries the following virulence genes: hblA, hblC, hblD, nheA, nheB, nheC and cytK; its tolerance Antibiotics included: ampicillin, amoxicillin-clavulanate, penicillin, cefotaxime, cefoxitin, co-trimoxazole, rifampicin, dalfopristin, and nitrofurantoin.
6. The standard strain of Bacillus cereus according to claim 3, wherein the strain 1761-2A carries the following virulence genes: nheA, nheB and nheC; antibiotics resistant to it include: ampicillin, Moxicillin-clavulanate, penicillin, cefotaxime, cefoxitin, tetracycline, and rifampicin.
7. The standard strain of Bacillus cereus according to claim 3, wherein the strain Y1712 carries the following virulence genes: nheA and cytK; antibiotics resistant to it include: ampicillin, amoxicillin-clar Retinoic acid, penicillin, cefotaxime, cefoxitin, tetracycline, and rifampicin.
8. The Bacillus cereus standard strain of claim 3, wherein the strain 2841-1B carries the following virulence genes: hblA, hblC, hblD, nheA, nheB, nheC, cytK and cesB; its Antibiotics tolerated include: ampicillin, cefotaxime, cefoxitin, penicillin, and rifampicin.
9. The standard strain of Bacillus cereus as claimed in claim 3, wherein the storage number of the strain 2801 is GDMCC 60865; the storage number of the strain 260-1B is: GDMCC 60867; the storage number of the strain 1761-2A The deposit number is GDMCC 60868; the deposit number of the strain Y1712 is GDMCC 60869; the deposit number of the strain 2841-1B is GDMCC 60866.
10. A freeze-drying protective agent for Bacillus cereus, characterized in that it comprises the following components by weight: 7 parts of cellobiose, 5 parts of skim milk powder, 2 parts of polyvinylpyrrolidone, 0.5 part of sodium glycerophosphate, phytose 1 part phosphoric acid.

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