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Method for diagnosing sporangium and egg capsule molecules of sarcocyst

A technology for sarcocystis and molecular diagnosis, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, microbiological measurement/inspection, etc. It can solve the problems of scarcity of samples and inability to use clinical diagnosis of human infectious diseases

Inactive Publication Date: 2007-12-26
KUNMING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Obviously, this method cannot be applied to the clinical diagnosis of human infectious diseases
2006, Elsheikha HM, Murphy AJ, Trembley SJ, Mansfield LS, Ghanam MS, el-Garhy MF. 2006 Molecular and microscopic techniques for detection of Sarcocystis neuronasporocysts in fecal samples. J Egypt Soc Parasitol. Aug;36(2):713- 25. This research focuses on a species of worm. The oocysts and sporocysts collected from the feces of experimentally infected animals are then amplified by PCR. Experimentally infected animals often have a large amount of infection and a large amount of material can be obtained. However, the human body or The number of samples in feces after natural infection of animals is extremely rare, and the report does not involve species identification

Method used

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  • Method for diagnosing sporangium and egg capsule molecules of sarcocyst

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Digestion analysis 1 (PCR-RFLP)

[0041] 1.1. Stool examination and collection of sporocysts and oocysts, including preparation of 37% ZnSO 4 Zinc sulfate floating solution (ZuSO 47Rz03319 plus distilled water 1000ml), take 1g of feces and add 37% ZnSO 4 Add 100ml of zinc sulfate floating liquid into a 15ml centrifuge tube. Set the centrifuge tube down. Use a pipette to suck up a few drops of the uppermost floating liquid in the centrifuge tube and transfer it to a glass slide, and examine it with a microscope at 150 times. Stick the sporangia and oocysts you see with clean paper. Put the disc into a 1.5ml centrifuge tube and store at -20°C.

[0042] 1.2. Obtain the target DNA fragment by PCR amplification

[0043] 1.2.1 PCR amplification conditions

[0044] 1.2.1.1 For each 50ul reaction volume, there are 5ul of 10×Buffer, 2ul of dNTPs, 2ul of BSA, 12ul of primers, 22ul of primers, 1.25U of Taq enzyme, 2ul of total DNA, and the balance is deionized water.

[00...

Embodiment 2

[0060] Digestion Analysis 2 (PCR-RFLP)

[0061] 2.1 Stool examination and collection of sporangia and oocysts, including preparation of 37% ZnSO 4 Zinc sulfate floating solution (ZuSO 4 7Hz03319 plus distilled water 1000ml), take 1g of feces and add 37% ZnSO 4 Add 100ml of zinc sulfate floating solution into a 15ml centrifuge tube. Set the centrifuge tube down. Use a pipette to suck up a few drops of the uppermost floating liquid in the centrifuge tube and transfer it to a glass slide, and examine it under a microscope at 130 times. Stick the sporangia and oocysts you see with clean paper. Put the disc into a 1.5ml centrifuge tube and store at -20°C.

[0062] 2.2 Obtain the target DNA fragment by PCR amplification

[0063] 2.2.1 PCR amplification conditions

[0064] 2.2.1.1 For each 50ul reaction volume, there are 5ul of 10×Buffer, 2ul of dNTPs, 2ul of BSA, 12ul of primers, 22ul of primers, 1.25U of Taq enzyme, 2ul of total DNA, and the balance is deionized water.

[0...

Embodiment 3

[0073] Sequencing was performed using the PCR and sequencing primers used in Table 1 for the study of the 18S rRNA gene of Sarcocystis sarcoides.

[0074] 3.1 Stool examination and collection of sporangia and oocysts, including preparation of 37% ZnSO 4 Zinc sulfate floating solution (ZuSO 4 7Hz03319 plus distilled water 1000ml), take 1g of feces and add 37% ZnSO 4 Add 100ml of zinc sulfate floating liquid into a 15ml centrifuge tube. Set the centrifuge tube down. Use a pipette to suck up a few drops of the uppermost floating liquid in the centrifuge tube and transfer it to a glass slide, and examine it with a microscope at 200 times. Stick the sporangia and oocysts you see with clean paper. Put the paper into a 1.5ml centrifuge tube and store at -20°C.

[0075] 3.2 Obtain the target DNA fragment by PCR amplification

[0076] 3.2.1 PCR amplification conditions

[0077] 3.2.1.1 For every 50ul reaction volume, there are 5ul of 10×Buffer, 2ul of dNTPs, 2ul of BSA, 12ul of ...

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Abstract

This invention relates to a method for detecting sporangium and oocyst of Sarcocystis. The method comprises: (1) mixing a small amount of feces with 37% ZnSO4 solution, centrifuging and standing; (2) detecting floating feces with a microscope, collecting sporangium and oocyst, placing in centrifugal tube, and storing at a low temperature; (3) performing PCR with the primers to amplify 18SrRNA in sporangium and oocyst of Sarcocystis; (4) digesting PCR product with restriction endonuclease, and detecting through gel electrophoresis, or detecting the nucleotide sequence with the primers used before. The primers can also be used as probes in hybridization reactions, or for manufacturing gene chips or microarrays. The method can be used for identifying Sarcocystis with high sensitivity and high specificity.

Description

technical field [0001] The invention belongs to the diagnosis of parasitic diseases, in particular to a method for diagnosing coccidiosis and coccidiosis of livestock, poultry and human body by using genes. Background technique [0002] Definitions of terms in the following: [0003] Oocyst: The stage at which Sarcocystis is detected in feces. One oocyst contains two sporangia. [0004] Sporangia: Mature sporangia contain 4 sporozoites. [0005] Cyst: Sarcocystis parasitizes in the muscle and is fusiform, and mature cysts contain bradyzoites. [0006] The parasites of the Sarcocystis genus are obligate intracellular parasitic protozoa, and their life cycle is typical of Apicomplexa, including schizotyposis, gametogenesis and sporulation. Sarcocystis is an obligate heterohost parasite, that is, it has to develop in the intermediate and terminal hosts respectively to complete its life cycle. Sarcocystis is widely parasitic on fish, reptiles, birds and mammals. Because man...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/25C12N15/30G01N33/50
Inventor 杨照青向征左仰贤张亚平陈新文周本江雷霖
Owner KUNMING MEDICAL UNIVERSITY
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