Method for detecting and counting live microbes

A technology of microorganisms and target microorganisms, which is applied in the field of inspection and counting of living microorganisms, can solve the problems of poor specificity of inspection and counting methods, time-consuming and laborious identification, etc., and achieve the effect of strengthening the detection of probiotics, short operation time and good reliability

Active Publication Date: 2011-01-05
BRIGHT DAIRY & FOOD CO LTD
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  • Claims
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AI Technical Summary

Problems solved by technology

[0012] Therefore, the technical problem to be solved in the present invention is to provide a new method for checking and counting living microorganisms for the poor specificity of traditional living microorganism detection and counting methods and the time-consuming and laborious defects of identification. Short time, strong specificity, accurate and reliable results

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  • Method for detecting and counting live microbes
  • Method for detecting and counting live microbes
  • Method for detecting and counting live microbes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Bifidobacterium longum (Bifidobacterium longum) count in embodiment 1 bifidobacterium yoghurt

[0034] 1. Design of Bifidobacterium longum qualitative PCR reagent

[0035] Sequence the gene sequence of the region between 16S rRNA and 23S rRNA of the fermented Bifidobacterium longum strain, compare and analyze on NCBI online, design Bifidobacterium longum qualitative PCR primers, upstream primers: 5'-CCA TCA TCC GCT TTC G-3', see SEQ ID NO.1 in the sequence listing; downstream primer: 5'-TGG CAG ACA GGA CCG ATG-3', see SEQ ID NO.2 in the sequence listing. The PCR product length is expected to be 215bp.

[0036] The bacterial positive marker PCR primers are 16S rRNA universal primers, upstream primers: 5'-AGA GTTTGA TCC TGG CTC AG-3'; downstream primers: 5'-C TGC TGC CTC CCG TAGGAG-3'. The length of the PCR product is expected to be about 300bp.

[0037] 2. Plate counting, PCR identification of colonies.

[0038] Aseptically weigh 25g of bifidobacterium yogurt (Changy...

Embodiment 2

[0046] Bifidobacterium longum live bacteria count in the intestinal tract of embodiment 2

[0047] 1. Bifidobacterium longum qualitative PCR reagent

[0048] With embodiment 1.

[0049] 2. Plate counting, PCR identification of colonies

[0050] Aseptically weigh fresh human feces, blow in carbon dioxide gas, oscillate and mix with sterile water with glass beads, dilute, and count on plates with TPY agar medium, the counting result is 6.3×10 9 cfu / g. Other steps are with embodiment 1.

[0051] The agarose electrophoresis diagram of the PCR product is shown in figure 2 , the upper band in the figure is the qualitative PCR band of Bifidobacterium longum with the corresponding number, and the lower part is the PCR band of the positive marker of the bacteria with the corresponding number, the number M is Wide Range DNA Marker, the number 0 is the negative control, and the number + is positive control, figure 2 Among them, 1, 2, 3...19 represent the selected single colonies ...

Embodiment 3

[0054] Detection and counting of common heat-resistant bacillus in embodiment 3 milk

[0055] Bacillus subtilis, B. licheniformis and B. pumilus in group Bacillus subtilis are common heat-resistant bacillus in milk, and they affect the quality of milk products. The specific multiplex PCR primer sequence comes from the literature "Application of Multiplex PCR to Identify Bacillus Commonly Used in Microbial Fertilizers" (Cao Fengming, Shen Delong, Li Jun, etc. Acta Microbiology, 2008, 48(5): 651-656), see Table 1; Bacteria positive Marker PCR primers are the same as in Example 1.

[0056] Table 1. PCR Primers

[0057]

[0058] Fresh milk extruded from cows that has just been purchased is treated at 85°C for 10 minutes to kill non-heat-resistant bacteria and enrich heat-resistant bacillus. Use pre-sterilized saline test tubes to make 10-fold serial dilutions, and the selected dilution factor is 10 1 、102 、10 3 Take 1ml of the test tube and put it into a sterile petri dish,...

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Abstract

The invention discloses a method for detecting and counting live microbes, comprising the following steps of: carrying out live bacteria cultivation and counting on a sample to be detected, and initially screening positive suspected microbes; (2) randomly selecting the positive suspected microbes obtained from the step (1), extracting genome DNA, carrying out PCR (Polymerase Chain Reaction) detection by using a specific primer of a target microbe, and counting. The invention organically combines a PCR technology with the traditional microbe counting technology, has high specificity and sensitivity and good reliability, can distinguish the live microbes and dead microbes which are contained in the sample and has little result error; and in addition, the invention has the advantages of short operating time, simple steps, nontoxic and innocuous reagent and high actual popularization and application prospect, is suitable for probiotics counting in mixed cultivation and planting counting research on probiotics in intestinal canals and is particularly suitable for accurate counting when disturbing microbes contained in the sample and target microbes to be detected are similar.

Description

technical field [0001] The invention belongs to the field of food microorganism detection, in particular to a method for detecting and counting living microorganisms. Background technique [0002] In food production, it is often necessary to detect the number of living organisms of specific microorganisms in samples. Different foods require different types and quantities of microorganisms to be detected, and there are relevant regulations in various countries. It is generally required that the number of live probiotics in the product reaches 1×10 6 cfu / g, the requirement of harmful microorganisms cannot be higher than the specified value to be qualified, and the number of living microorganisms in the product directly affects the quality of the product. [0003] At present, the commonly used microbial living counting methods in my country (such as: GB / T4789134-2003 and GB / T4789135-2003 detect and count bifidobacteria and lactic acid bacteria respectively) include plate count...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11
Inventor 张红发郭本恒王荫榆舒妹
Owner BRIGHT DAIRY & FOOD CO LTD
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