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CN-122012491-A - Method for extracting giardia and cryptosporidium nucleic acid and application thereof

CN122012491ACN 122012491 ACN122012491 ACN 122012491ACN-122012491-A

Abstract

The invention discloses a method for extracting giardia and cryptosporidium nucleic acid and application thereof. The method comprises the steps of mixing a sample containing giardia and/or cryptosporidium with lysozyme and proteinase K, incubating and digesting, adding a lysis enhancing liquid and glass beads into the digested sample, performing ultrasonic treatment, centrifuging to obtain a supernatant, and performing automatic nucleic acid extraction and purification on the obtained supernatant. The invention shortens the sample processing time to within 3 h, greatly improves the detection efficiency, reduces the detection cost, and has excellent cracking effect, high sensitivity and strong specificity.

Inventors

  • LIU YANHONG
  • SU SHENGBING
  • Gu Saiyi
  • YING QINGJIE
  • ZHANG HUIYANG

Assignees

  • 江苏奇天基因生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260225

Claims (10)

  1. 1. A method for extracting giardia and/or cryptosporidium nucleic acid is characterized by comprising the steps of mixing a sample containing giardia and/or cryptosporidium with lysozyme and proteinase K, incubating and digesting, adding a lysis enhancing liquid and glass beads into the digested sample, performing ultrasonic treatment, centrifuging to obtain a supernatant, and performing automatic nucleic acid extraction and purification on the obtained supernatant.
  2. 2. The method according to claim 1, characterized in that it comprises the steps of: (1) Sample pretreatment, namely placing a sample containing giardia and/or cryptosporidium into a centrifuge tube; (2) Enzyme digestion, namely adding lysozyme and proteinase K into the centrifuge tube in the step (1), uniformly mixing, and incubating for digestion; (3) Ultrasonic crushing, namely adding a cracking enhancement solution and glass beads into the digested sample in the step (2), performing ultrasonic treatment, and centrifuging after ultrasonic treatment to obtain a supernatant; (4) Nucleic acid extraction, namely, the obtained supernatant is subjected to automatic nucleic acid extraction and purification by using a magnetic bead method.
  3. 3. The method according to claim 2, wherein the volume ratio of lysozyme to proteinase K in step (2) is 1mL (30-80) μl; preferably, the temperature of the incubation digest in step (2) is 40-60 ℃ and the time of the incubation digest is 55-75 min.
  4. 4. A method according to claim 2 or 3, wherein the time of the ultrasound in step (3) is 10-25 min.
  5. 5. The method according to any one of claims 2 to 4, wherein the centrifugation in step (3) is performed at a rotational speed of 10000 to 30000 rpm for a time of 2 to 5 min.
  6. 6. The method according to any one of claims 2 to 5, wherein the automated nucleic acid extraction and purification of the supernatant obtained in step (4) using the magnetic bead method comprises the steps of: (a) Combining, namely mixing supernatant obtained by centrifugation with magnetic beads for 1-2 min, and magnetically attracting the magnetic beads for 30-90 min; (b) Cracking, namely heating and cracking 15-25 min under the condition of 85-95 ℃ and magnetically attracting 100-150 min by using magnetic beads; (c) Washing, namely mixing the washed liquid 1 with 1-2 min and magnetic beads of 100-150 min after cracking, mixing the washed liquid 2 with 1-2 min and magnetic beads of 100-150 min, and mixing the washed liquid 3 with 1-2 min and magnetic beads of 100-150 min; (d) Eluting by eluting with eluent at 80-90deg.C for 5-10 min, air drying for 5-10 min, magnetically absorbing with magnetic beads for 30-90 min, and discarding magnetic beads to obtain solution containing giardia and/or Cryptosporidium nucleic acid; preferably, the washing liquid 1 comprises any one or a combination of at least two of guanidine hydrochloride, guanidine isothiocyanate or a detergent; Preferably, the washing liquid 2 comprises any one or a combination of at least two of sodium chloride solution, ethanol-isopropanol or Tris; Preferably, the washing liquid 3 comprises acetone; preferably, the eluent comprises EDTA and/or Tris.
  7. 7. Use of the method of any one of claims 1-6 for the preparation of a product for detecting giardia and/or cryptosporidium nucleic acids in tap water.
  8. 8. Use of the method of any one of claims 1-6 for monitoring the water quality safety of tap water.
  9. 9. Use of the method of any one of claims 1-6 for the preparation of an in vitro diagnostic reagent for the diagnosis of giardia and/or cryptosporidium infection.
  10. 10. A method for detecting giardia and/or cryptosporidium nucleic acid in tap water, which is characterized in that the method comprises the steps of extracting giardia and/or cryptosporidium nucleic acid by using the method of any one of claims 1-6, and performing amplification detection on the extracted nucleic acid by using a isothermal amplification technology.

Description

Method for extracting giardia and cryptosporidium nucleic acid and application thereof Technical Field The invention belongs to the technical field of environmental monitoring, in-vitro diagnosis and biological prevention and control, and relates to a method for extracting giardia and cryptosporidium nucleic acid and application thereof. Background Giardia belongs to the class of dinoflagellates, and is a protozoa parasitic to the human intestinal tract, and mainly has two forms, namely a trophozoite with exercise and ingestion functions and a capsule with a protective shell, the capsule has strong resistance to the external environment, can survive for months in cold water, and the conventional chlorine disinfection (such as tap water disinfection) has poor effect on the giardia. Pathogens are transmitted primarily through the faecal-oral route. Cryptosporidium belongs to Acremodelling, has relatively close relation with plasmodium and toxoplasma, and has oocysts as infection stage and infectious effect during discharge. Pathogens are transmitted primarily through the faecal-oral route, with oocysts expelled by infected or sick animals (e.g., cattle), contaminating water sources, food, or through direct contact (e.g., patient care, diaper change). But also through swimming pools. Giardia has a tough encapsulating wall structure (mainly composed of chitin, protein and carbohydrate), and the core of nucleic acid cleavage is to effectively destroy the encapsulating wall and the cell membrane of the worm body while avoiding degradation of the nucleic acid. The method has the core difficulty that the encapsulation wall is tough and resistant to chemical/physical damage, the insect body needs to be preferentially damaged to release the insect body, the insect body cell membrane contains lipid and protein, further cracking is needed to release nucleic acid, nuclease (endogenous/exogenous) is prevented from degrading DNA/RNA in the cracking process, inhibitors (such as EDTA and RNase inhibitors) need to be synchronously added, and the method has good requirements on tolerance of a detection system. Common nucleic acid cleavage methods are mainly physical and chemical cleavage. The physical lysis has the defects of low lysis efficiency, easy RNA degradation (strict temperature control is needed), high sample treatment difficulty and high instrument requirement, and is not suitable for large-batch and mobile detection requirements. The chemical cracking has the defects of quick cracking, avoidance of nucleic acid degradation, poor cracking effect, complicated sample treatment flow, high pollution of chemical reagents, particularly high guanidine salt toxicity, consideration of the influence of operators and environmental pollution, and easiness in causing serious interference on detection sensitivity and specificity. Therefore, it is needed to provide an extraction method which can efficiently and synchronously break Jie Gudi flagellate and cryptosporidium tough capsule walls, realize rapid and stable release of nucleic acid, is suitable for automation and is suitable for mass screening of water quality. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the invention provides a method for extracting giardia and cryptosporidium nucleic acid and application thereof, shortens the detection time, improves the detection sensitivity and stability, and provides reliable technical support for drinking water safety monitoring. In order to achieve the aim of the invention, the invention adopts the following technical scheme: In a first aspect, the invention provides a method for extracting giardia and/or cryptosporidium nucleic acid, the method comprising mixing a sample containing giardia and/or cryptosporidium with lysozyme and proteinase K, incubating for digestion, adding a lysis enhancing liquid and glass beads to the digested sample, performing ultrasonic treatment, centrifuging after ultrasonic treatment to obtain a supernatant, and performing automatic nucleic acid extraction and purification on the obtained supernatant. The invention adopts a two-step pretreatment strategy of 'enzymatic digestion-ultrasonic collaborative breaking', shortens the sample treatment time to within 3 h, greatly improves the detection efficiency, reduces the detection cost, and has good cracking effect, high sensitivity and strong specificity. Preferably, the method comprises the steps of: (1) Sample pretreatment, namely placing a sample containing giardia and/or cryptosporidium into a centrifuge tube; (2) Enzyme digestion, namely adding lysozyme and proteinase K into the centrifuge tube in the step (1), uniformly mixing, and incubating for digestion; (3) Ultrasonic crushing, namely adding a cracking enhancement solution and glass beads into the digested sample in the step (2), performing ultrasonic treatment, and centrifuging after ultrasonic treatment to obtain a supernatant; (4) Nucleic acid extraction, n