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Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit

A detection kit, staphylococcus technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of time-consuming instability, low sensitivity, poor specificity, etc., to improve quarantine level, the effect of overcoming low sensitivity and simplifying the detection procedure

Inactive Publication Date: 2012-01-04
刘箐 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to overcome the deficiencies of low sensitivity, poor specificity, and time-consuming and unstable detection methods of existing food-borne pathogenic bacteria, the method of effectively combining pathogen enrichment and gene level detection uses immunological methods to first enrich the pathogens in the sample. Set, and then carry out PCR amplification, which can greatly improve the detection sensitivity, and at the same time greatly improve the detection efficiency

Method used

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  • Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit
  • Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit
  • Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 A kind of Staphylococcus aureus immunocapture PCR detection kit includes:

[0055] 10× PCR buffer 5uL, dNTPs 2.5mmo l / L 2uL, Taq DNA polymerase 5U / L 0.25uL, forward primer 10p mol l / L 2uL, primer 5'-GCGATTGATGGTGATACGGTT-3'; reverse primer 10p mo l / L 2uL, primer 5'-AGCCAAGCCTTGACGAACTAAAGC-3'; double distilled water 99%~100%; 3 detection PCR tubes coated with Staphylococcus aureus polyclonal antibody; coated with Staphylococcus aureus polyclonal antibody 3 positive control PCR tubes (inactivated Staphylococcus aureus liquid) tubes; 3 negative control PCR tubes (sterilized PBS) tubes coated with Staphylococcus aureus polyclonal antibody; 1 positive control tube; Negative control 1 tube.

Embodiment 2

[0056] Embodiment 2 A kind of using method of Staphylococcus aureus direct PCR detection kit, its main feature is that the steps are:

[0057] After the standard bacteria were cultured for 18 hours, take 1ml of the bacterial solution and dilute it 10-fold in sequence into sterile PBS, and mark it as 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 , and then take 10 -5 、10 -6 、10 -7 Each 100ul of the gradient pure bacterial solution was placed in a petri dish for plate colony counting. The concentration is: 2.35×10 8 -2.35×10 1 CFU / ml.

[0058] Take 5 ul of gradient bacterial liquid homogenate in a PCR tube, add 37.25 ul of deionized water, and run on a PCR instrument at 99 0 C cleavage for 10min, then quickly placed on ice to cool for 5min, after centrifugation, add 5ul of 10×PCR buffer, 4ul of 2.5uMdNTPs, 0.25ul of 5U / ul Taq DNA polymerase, 2ul of upstream primer 5'-GCGATTGATGGTGATACGGTT-3' 8-10 pmol / μL; 2ul primer 5'-AGCCAAGCCTTGACGAACTAAAGC-3' 8-10 pmol / μL; th...

Embodiment 3

[0060] Example 3: Detection of Immunocapture PCR Specificity, see Figure 5 ,

[0061] Tested strains: Staphylococcus aureus ATCC 27660, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 43251, Listeria monocytogenes ATCC 7644, Enterobacter sakazakii ATCC 29004, Enterobacter sakazakii ATCC 29554, Salmonella Enteritidis ATCC 13076, Yersinia enterocolitica ATCC 23715, Vibrio parahaemolyticus ATCC 17802, Bacillus cereus ATCC 11778, negative control.

[0062] The test results showed that among the above ten tested bacteria, only Staphylococcus aureus detected the target band, and none of the others detected the target band, indicating that the kit has relatively good specificity.

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Abstract

The invention relates to a gene detection kit of staphylococcus aureus, and the detection kit belongs to the field of detection of food-borne pathogenic bacteria and is specially used for detecting the staphylococcus aureus (SA). The immunocapture PCR (polymerase chain reaction) detection kit of the staphylococcus aureus is mainly characterized by comprising 5 mu L of 10*PCR buffer, 2 mu L of dNTPs (deoxyribonucleoside triphosphates) (2.5 mmol / L), 0.25 mu L of Taq DNA (deoxyribonucleic acid) polymerase (5 U / L), 2 mu L of forward primer (10 pmol / L) which refers to primer 5'-GCGATTGATGGTGATACGGTT-3', 2 mu L of reverse primer (10 pmol / L) which refers to primer 5'-AGCCAAGCCTTGACGAACTAAAGC-3' and 99%-100% of double-distilled water, as well as a detection PCR tube coated with a staphylococcus aureus polyclonal antibody, a positive control PCR tube coated with the staphylococcus aureus polyclonal antibody, namely inactivated staphylococcus aureus bacteria liquid, a negative control PCR tube coated with the staphylococcus aureus polyclonal antibody, namely sterilized PBS (phosphate-buffered solution), a positive control 1 tube and a negative control 1 tube. The immunocapture PCR detection kit has the advantages of being capable of quickly, simply and accurately detecting the pathogenic bacteria.

Description

technical field [0001] The invention is a gene detection kit for Staphylococcus aureus, which belongs to the field of detection of food-borne pathogenic bacteria, and is specifically aimed at Staphylococcus aureus ( staphylococcus aureus, SA ) detection. It is suitable for the rapid detection of Staphylococcus aureus in food. Background technique [0002] Staphylococcus aureus ( Staphyloccocus aureus , SA) is an important human pathogen belonging to the Staphylococcus genus, which can cause a variety of serious infections. There is another name for "flesh-loving bacteria". Typical Staphylococcus aureus is spherical, with a diameter of about 0.8 μm, arranged in clusters of grapes under the microscope. Staphylococcus aureus has no spores, flagella, most of them have no capsule, and Gram stain is positive. Staphylococcus aureus has low nutritional requirements and grows well on ordinary medium, aerobic or facultative anaerobic, the optimum growth temperature is 37°C, the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/10C12R1/445
Inventor 夏俊芳刘箐
Owner 刘箐
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