Polymerase chain reaction (PCR) method for identifying four pathogens in prenatal and postnatal care examination through single tube and kit thereof

A single, reaction tube technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of repeated fragments, GC content deviation, etc.

Active Publication Date: 2014-10-29
苏州华益美生物科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene sequences of pathogens such as toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus have the characteristics of high fragment repetition, GC content deviation, and few specific short fragments, while the specific long fragments interfere seriously with each other. Up to now, it is impossible to pass the PCR method in a single tube for one-time detection. Only a chip hybridization detection method such as Chinese Patent (Application) No. 201410016220 has been developed, in which the probes are separated and spotted to avoid mutual interference.

Method used

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  • Polymerase chain reaction (PCR) method for identifying four pathogens in prenatal and postnatal care examination through single tube and kit thereof

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Experimental program
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Effect test

Embodiment 1

[0085] 1. Nucleic acid extraction

[0086] According to the virus storage and research specifications of the Ministry of Health, nucleic acid extraction was performed on Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus type II strains.

[0087] The extraction of nucleic acid is carried out according to the conventional magnetic bead extraction method, and the method is as follows: add 10 μl standard sample (pseudovirus particle 1*10 4IU / ml) was added to 90 μl nucleic acid extraction solution (formulation and final concentration: guanidine isothiocyanate 1.2M, sodium edetate (pH8.0) 10mM, Tween-202% (W / W), perchloric acid NaOH 1.0M, ethanol 40% (V / V), Tris-HCl (pH8.0) 10mM), incubate at 42°C for 10min, then add 10μl Magnetic bead suspension (50 mg / mL, purchased from Beijing Aibigen Biotechnology Co., Ltd.), after oscillating and mixing evenly, put it on a magnetic rack to apply a magnetic field, discard the liquid, and then add 200 μl of washing sol...

Embodiment 2

[0137] Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus type II, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Human papillomavirus and other samples were used as templates for this experiment The designed primers and probes were used for real-time fluorescent PCR reaction. As a result, only Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus II produced fluorescent signals. See Table 1 for the simultaneous PCR amplification curves of various viruses in a single tube. See attached for example figure 1 . This shows that the method is specific for the detection of Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus II.

[0138] Table 1 Pathogen method-specific fluorescent PCR amplification results in single-tube detection of four types of prenatal and postnatal care inspections

[0139] sample name

[0140]

[0141] Th...

Embodiment 3

[0143] As of this patent application, we have tested the samples of 78 subjects one by one with the above method, and at the same time, we have performed separate PCR amplification detection and Western blot detection of different viruses on the same sample (a total of 8 verifications, if Result has conflict, repeat 3 times to determine result again, take majority result as the criterion) as verification, use method of the present invention to find no false-negative and false-positive detection result.

[0144] Therefore, the present invention adopts the fluorescent PCR method to detect four kinds of pathogens in prenatal and postnatal care inspections in a single tube, and can be completely used in a fully automatic fluorescent PCR instrument, which has the characteristics of easy operation, high sensitivity, good accuracy and repeatability. Therefore, the invention can be used for fast and large amount of in vitro sample detection, and has good application prospects.

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Abstract

The invention discloses a real-time fluorescent polymerase chain reaction (PCR) method for simultaneously detecting four targeting nucleic acids in a single PCR reaction tube. The method is used for detecting toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus in samples. The method is fast in operation, is finished through one step and is high in accuracy and sensitivity, and false positive and false negative results are not discovered in practice detection. In addition, the invention also relates to a reagent involved in the PCR method, as well as a detection kit for the method and preparation and application of the detection kit.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection. Specifically, the invention provides a fluorescent PCR method and a kit thereof for rapidly and parallelly detecting four pathogens (toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus) in the same PCR tube . Background technique [0002] Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus are four common pathogens in prenatal and postnatal care inspections in my country. [0003] Toxoplasma gondii is called three corpses in traditional Chinese medicine, which 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 can be contracted by eating undercooked contaminated meat, or by keeping pets. After a person is infected, he suffers from toxoplasmosis. Once a pregnant woman i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2561/113C12Q2563/107
Inventor 齐洁婷刘劲林陈红干张必新王川陈悦科郭志武
Owner 苏州华益美生物科技有限公司
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