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Quality evaluation process of inactivate effect of methylen blue photochemical virus and quality-controlling products thereof

A technology for virus inactivation and quality evaluation, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, DNA/RNA fragments, etc., can solve the problems of long experimental period, cumbersome operation, expensive price, etc. Short, easy to operate, good accuracy

Active Publication Date: 2011-01-12
SHANGHAI BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the in vitro culture method has strict requirements on the experimental conditions, cell culture and virus preservation are under standardized control, the operation is cumbersome, and the experiment cycle is long, which is not conducive to carrying out this work in grassroots units
In addition, although the animal model method has advantages in evaluating virus infectivity from the overall level of the organism, it is expensive and has a long experimental cycle, which is only suitable for use in the research and development stage, and is not suitable for routine work.

Method used

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  • Quality evaluation process of inactivate effect of methylen blue photochemical virus and quality-controlling products thereof
  • Quality evaluation process of inactivate effect of methylen blue photochemical virus and quality-controlling products thereof
  • Quality evaluation process of inactivate effect of methylen blue photochemical virus and quality-controlling products thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] 1. Cloning establishment:

[0080] According to the HCV1b type sequence (AY460204) in GenBank, PCR primers covering HCV 5'-NCR and core region fragments were designed using Gene runner software. The sequence is: 5'-CGGGATCC CGGAAGATAGAGAAAGAGCAACCG-3' (SEQ ID NO: 4), the length of the amplified fragment is 844bp, and the primers are synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. After extracting HCV RNA in plasma (Shanghai Blood Center) of blood donors who were positive for both anti-HCV and HCV RNA, Trizol (Gibco BRL) reagent was used, and RNA PCR KIT (Takara Company) was used for reverse transcription and PCR reactions. The reaction parameters were: reverse transcription at 42°C for 30 minutes, pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, with 30 cycles of denaturation, annealing, and extension. After the PCR product was purified, it was c...

Embodiment 2

[0097] 1. Preparation of experimental SV and preparation of virus-containing plasma:

[0098] (1) Preparation of SV for experiment

[0099] SV was provided by the Institute of Virology, Chinese Academy of Preventive Medicine, and BHK cells were used as hosts for 3 generations. Collect the supernatant and measure the virus titer>8.5lgTCID 50 / ml, aliquoted, stored at -80°C, and used for later use.

[0100] (2) Preparation of virus-containing plasma:

[0101] In the PVC plastic bag containing 100ml anticoagulant plasma, inoculate the virus according to the capacity ratio of plasma: virus solution=9:1, and mix well.

[0102] 2. Virus inactivation experiment:

[0103] In the above-mentioned virus-containing plasma / solution, add methylene blue (MB) so that the final concentrations are 0.5 μmol / L, 1.0 μmol / L, and 1.5 μmol / L, and then irradiate with 30000 Lux visible light, and the irradiation time is 0′( not illuminated), 30', 60'.

[0104] 3. Viral nucleic acid detection (qua...

Embodiment 3

[0114] 1. The inactivated product of step 2 in Example 2 is sampled respectively, inoculated with BHK cells, and measured by cytopathic method ("Medical Experimental Virology", People's Military Medical Publishing House (first edition) P102-109 Du Ping). Infectious titer (TCID 50 / ml), the results are as follows:

[0115] Infectious inactivation results of SV at different concentrations and different contact times

[0116]

[0117] *Indicates that the method used has not detected the virus titer

[0118] The results confirmed that after inactivation, the virus infectivity disappeared.

[0119] 2. Result analysis: It can be seen from Example 2 that under MB-P treatment, SV virus RNA was degraded, and this experiment proved that after MB-P treatment, SV virus infectivity disappeared, thus confirming the viral nucleic acid There is a certain correlation between the degradation of the virus and the disappearance of the infectivity of the virus.

[0120] Example 4 Preparatio...

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Abstract

The invention discloses a novel evaluation and detection method for methylene blue photochemical viral inactivation effect quality, which relates to the blood virus inactivation effect quality evaluation field. The invention solves the problems that in the prior art, an MB-P inactivation effect quality evaluation method has high cost and long period, and is not suitable for being developed in regular work. The invention comprises the following steps that: while the inactivation is performed to blood or blood products, quality control products are inactivated under the same conditions; the quality control products contain recombinant particles of HCV virus genes or contain RNA fragments of HCV virus gene vitro transcription; a PCR technology is utilized to augment special areas of the HCV virus genes in the quality control products through a special primer; and the quality evaluation is made to the methylene blue photochemical viral inactivation effect from molecular level by monitoring the PRC result. The invention also discloses a quality control product thereof. The method has simple operation, good accuracy and short experimental period, is easy to achieve the needed experimental conditions, and is more economic and effective.

Description

technical field [0001] The invention relates to a quality evaluation method for virus inactivation effects of blood and blood components, and establishment of corresponding quality control products, in particular to a quality evaluation method for methylene blue photochemical virus inactivation effects and quality control products. Background technique [0002] With the development of medical science, more and more attention has been paid to the safety of blood and blood products. Blood safety not only depends on reasonable screening of blood donors, strict virus screening, but also on safe and effective virus inactivation technology. It is an important guarantee for safe blood transfusion. Methylene blue (Methylene blue, MB) photochemical method is considered to be a very promising method for inactivating viruses in clinical single-serving plasma. Since the virus inactivation technology is affected by various conditions and factors, such as the concentration of MB, light i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11A61K35/14C12N15/63
Inventor 郑岚王迅黄宇闻莫琴刘晓颖钱开诚
Owner SHANGHAI BLOOD CENT
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