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Universal type fluorescent quantitative PCR method for detecting platelet bacterial pollution

A fluorescence quantitative and detection method technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of complex result interpretation standards, poor sensitivity, long detection time, etc., to achieve effective auxiliary diagnostic methods and solve bacteria Whether or not, the effect of reliable detection requirements

Inactive Publication Date: 2015-02-25
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the bacterial culture detection method is used as the gold standard. Among them, the Bact / ALERT bacterial culture monitoring system and the PalleBDS bacterial detection system are certified by the FDA. 2 , accumulated CO 2 , nutrient composition changes, etc., this method takes a long time to detect, it takes 1 to 2 days, and there will be cases of missed detection.
In addition, the FDA-certified PGD test strip detection system uses monoclonal antibodies to bacterial surface-specific antigens to quickly detect bacterial contamination of platelet products at the bedside, but this method cannot be used as a quality control method for platelets, and its sensitivity is poor. Gram-negative bacteria are difficult to detect
The Scansystem system uses monoclonal antibodies to enrich platelets to filter samples, label the bacterial DNA with permeabilizers and fluorescent agents, and use laser scanning to determine the contamination. Although the detection time of this method is short, the sensitivity is not high. The final result Interpretation criteria are complex
At present, there are no detection reagents and instruments related to the contamination of platelet bacterial products in China.

Method used

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  • Universal type fluorescent quantitative PCR method for detecting platelet bacterial pollution
  • Universal type fluorescent quantitative PCR method for detecting platelet bacterial pollution
  • Universal type fluorescent quantitative PCR method for detecting platelet bacterial pollution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Specificity of CR5-F and CR7-R primers for bacterial and human genomes

[0059] Dry powder primers and probes were dissolved in TE buffer to 10 μM; human genomic DNA and bacterial genomic DNA were extracted with kits, and quantified with a microspectrophotometer; conventional PCR was used for amplification, and the 30 μL reaction system included:

[0060] 2X Master Mix PCR Mix Buffer 15 μL

[0061] CR5-F upstream primer (10μM) 1 μL

[0062] CR7-R downstream primer (10μM) 1 μL

[0063]Genome template (50~100ng) 1 μL

[0064] Make up to 12 μL with double distilled water.

[0065] Reaction conditions: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, 30 cycles; final incubation at 72 °C for 5 min. PCR amplification products were detected by agarose gel electrophoresis, and the results were recorded, such as Figure 4 It was shown that this pair of primers amplified only the bacteri...

Embodiment 2

[0066] Example 2: Fluorescence quantitative PCR detects bacterial DNA

[0067] The embodiment does not use platelet product samples for detection, but uses human whole blood genome and bacterial genome alone or mixed to simulate, with human genome as a negative control, and mixed gradient dilution of human genome as a detection sample. Fluorescence quantitative PCR is performed. Detection, 30μL reaction system, including:

[0068] 2X Master Mix PCR Mix Buffer 15 μL

[0069] CR5-F upstream primer (10μM) 1 μL

[0070] CR7-R downstream primer (10μM) 1 μL

[0071] CR6-probe probe (10 μM) 0.5 μL

[0072] Internal reference probe (10μM) 0.5 μL

[0073] Genome template (50~100ng) 1 μL

[0074] Artificial reference (diluted to about 100 copies) 1 μL

[0075] Make up to 10 μL with double distilled water.

[0076] Reaction conditions: pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 20 s, annealing, extension and fluorescence detection at 60 °C for 1 min, a total of...

Embodiment 3

[0077] Embodiment 3: Difference and interpretation between the detection limit of artificial internal reference and the Ct value of sample detection

[0078] In this example, the artificial internal reference is synthesized in an amount of about 33ug per OD of the gene, and the number of molecules of the artificial internal reference is 38881.9, which is converted into a copy number for gradient dilution, and can be determined by fluorescence quantitative PCR under the conditions of this example (including reagents, Consumables, equipment, operation and other factors), the minimum number of internal parameters that can be detected is 20 copies. The OD of cultured Escherichia coli was detected by visible light spectrophotometry 600 value, in OD 600 When =1.0, there are about 6.3x10 in 1mL culture medium 8 50 μL of the dilution solution was used for lysing at 95°C for 20 minutes. After centrifugation, 1 μL of the diluted solution was used as a template for fluorescence quantit...

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Abstract

The invention discloses a universal type fluorescent quantitative PCR method for detecting platelet bacterial pollution. The fluorescent quantitative PCR method employs a pair of universal type primers detecting bacteria, an artificial internal interference of the universal type primers, a first fluorescently-labeled fluorescent probe and a second fluorescently-labeled fluorescent probe; the two fluorescent probes employ different fluorescent labels; the first fluorescently-labeled fluorescent probe is a universal type fluorescent probe used for detecting bacteria, and the second fluorescently-labeled fluorescent probe is an internal reference probe used for detecting the artificial internal interference; and a routine PCR amplification technology is employed for detection. According to the above technical scheme, the detection method is suitable for screening on bacteria pollution of platelet products, is capable of fully solving the bacteria existence, bacteria content, bacteria survival state and other problems in bacteria pollution of the platelet products, satisfies rapid, accurate and reliable detection requirements, and provides a scientific effective auxiliary diagnosis method for safe infusion of platelet.

Description

technical field [0001] The invention relates to the field of molecular diagnosis and detection, in particular to a universal fluorescent quantitative PCR method for detecting bacterial contamination of platelets. Background technique [0002] The pathogens transmitted through blood transfusion are mainly viruses, bacteria and protozoa, etc. Currently, only hepatitis B, hepatitis C, HIV and Treponema pallidum are included in blood source screening in my country. Among the infectious risks of transfusion, bacterial contamination and septic transfusion reactions have not received sufficient attention. Platelet products must be stored with shaking at (22±2)℃. The probability of bacterial contamination in this environment is higher than the probability of bacterial contamination or viral infection of other blood components. Therefore, the effective shelf life of platelet products is very short. The general rule is 24 hours or 5 days. Studies have shown that bacterial contaminat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6851
Inventor 蒙青林李勇田晶晶魏双施王红梅段生宝丁少华陈晔洲李冬
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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