Toxoplasma detecting parallel probes, gene chip, kit and detection method

A technology for detecting gene chips and detection kits, which is applied in the field of molecular biology, can solve the problems of false positives, low sensitivity, and poor specificity, and achieve high specificity, simple detection operation, and improved accuracy and precision. Effect

Inactive Publication Date: 2016-09-07
重庆威斯腾生物医药科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current method for detecting Toxoplasma gondii is the ELISA method, which generally has the disadvantages of low sensitivity, poor specificity, and false positives.

Method used

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  • Toxoplasma detecting parallel probes, gene chip, kit and detection method
  • Toxoplasma detecting parallel probes, gene chip, kit and detection method
  • Toxoplasma detecting parallel probes, gene chip, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Target Fragment Primer and Probe Design

[0024] Look up the gene sequence of Toxoplasma gondii in the NCBI database, select a highly specific gene sequence as the target sequence (ID number: AF179871.1), and design primers and probes. After the target sequence is determined, according to the design principles of primers and probes, the amplification primers and probes of the target genes of Toxoplasma gondii are designed. In order to make the chip results can be directly interpreted by naked eyes after being developed with the chromogenic solution, the target genes of Toxoplasma gondii are amplified The 5' end of the R primer of the primer is labeled with biotin, and the specific sequence is as follows:

[0025] Toxoplasma gondii (TOX) target gene amplification primers:

[0026] Primer F (TOX-F): 5'-CGTATTGTCGAGTAGATCAG-3' (SEQ ID NO.4),

[0027] R primer (TOX-R): 5'-biotin-TCGCTGCGGA GACAGCGAAG (SEQ ID NO.5)-3'.

[0028] TOX parallel probe:

[0029] TOX ...

Embodiment 2

[0035] The preparation of embodiment 2 chip

[0036] The parallel probe of TOX among the embodiment 1 is spotted on the glass substrate of amino modification according to the order of table 1, obtains the gene chip that contains probe (such as figure 1 shown). The probe concentration was 30 μM, 0.2 μL per spot, and then incubated at 80° C. for 1.5 hours.

[0037] Table 1 Arrangement sequence of chip probes for Toxoplasma gondii detection

[0038]

Embodiment 3

[0039] Embodiment 3 detection method

[0040] 1. Genome extraction of samples to be tested

[0041] Use the virus genome DNA / RNA co-extraction kit and follow the steps in the operation manual to extract the genome of the sample to be tested as the amplification template.

[0042] 2. PCR amplification of the target fragment

[0043] After a lot of experiments and exploration, the target gene PCR amplification system and PCR reaction program for TOX detection were determined.

[0044] The PCR amplification system with a total volume of 10 μL contains the following reagents:

[0045] name

Amount added

Premix Taq

5μL

F primer (10 μM)

0.2 μL

R primer (10 μM)

0.2 μL

amplified template

0.4μL

double distilled water

4.2 μL

[0046] The PCR reaction program was: 95°C for 5 minutes; 95°C for 10s, 55°C for 30s, 72°C for 30s, 30 cycles; 72°C for 10min.

[0047] 3. Hybridization of PCR products with chips

[004...

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Abstract

The invention discloses toxoplasma detecting parallel probes. The toxoplasma detecting parallel probes comprise a probe 1, a probe 2 and a probe 3, with respective sequences as follows: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The invention further discloses a gene chip comprising the probes as well as a kit comprising the gene chip, and further discloses a method for detecting toxoplasma by using the kit. The method comprises the following steps: detecting by using the toxoplasma detecting kit provided by the invention, wherein when detection results displayed by the three probes of a detected object are consistent, whether a sample is infected by the toxoplasma can be judged, and when the detection results displayed by the three probes of the detected object are inconsistent, whether the sample to be detected is positive or negative cannot be judged; and redetecting by using the detecting kit or further verifying by using an existing other detection method. Through toxoplasma detecting parallel probes, the gene chip, the kit and the detection method, operation is simple, results are easy to read, needs of hospital detection, prenatal detection and other on-site detections are met, and the results are accurate and reliable.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a parallel probe for detecting toxoplasma gondii, a gene chip, a kit and a detection method. Background technique [0002] Toxoplasma (Toxoplasma) Chinese medicine called three corpses, belonging to the apical complex subphylum, Sporozoa, Eucoccidia, Isospora Coccidiaceae, Toxoplasma genus, is an intracellular parasite. Parasitic in the cells, with the blood flow, reach all parts of the body, damage the brain, heart, fundus, resulting in a decline in human immunity, suffering from various diseases. Toxoplasma gondii is a parasitic disease that can be transmitted through water sources and food sources. It has great potential danger to human and animal health. They parasitize in the host cells and can infect all warm-blooded vertebrates including humans. Toxoplasma infection in humans can be very serious and even life-threatening in some cases, such as fetuses during pregnancy and im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6888C12Q1/6837C12Q2600/158C12Q2565/501C12Q2531/113
Inventor 周勇徐建
Owner 重庆威斯腾生物医药科技有限责任公司
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