Method for extracting giardia and cryptosporidium nucleic acid and application thereof

Abstract

The invention discloses a method for extracting giardia and cryptosporidium nucleic acid and application thereof. The method comprises the following steps: mixing a sample containing giardia and / or cryptosporidium with lysozyme and protease K, and incubating and digesting; adding a lysis enhancement solution and glass beads into the digested sample, carrying out ultrasonic treatment, and centrifuging after ultrasonic treatment to obtain a supernatant; and carrying out automatic nucleic acid extraction and purification on the obtained supernate. According to the method, the sample treatment time is shortened to be within 3 h, the detection efficiency is greatly improved, the detection cost is reduced, the cracking effect is excellent, the sensitivity is high, and the specificity is high.

Description

Technical Field

[0001] This invention belongs to the fields of environmental monitoring, in vitro diagnostics, and biocontrol technology, and relates to a method for extracting nucleic acids from Giardia lamblia and Cryptosporidium and its application. Background Technology

[0002] Giardia lamblia, belonging to the class Ognathidae, is a protozoan that parasitizes the human intestine. It exists in two main forms: a trophozoite with motility and feeding capabilities, and a cyst with a protective exoskeleton. The cysts are highly resistant to environmental changes, surviving for months in cold water, and are poorly protected by conventional chlorination methods (such as tap water disinfection). The pathogen is primarily transmitted via the fecal-oral route. Cryptosporidium, belonging to the phylum Apicocytocomplexa, is closely related to Plasmodium and Toxoplasma gondii. Its oocysts are the infectious stage and are infectious upon excretion. The pathogen is primarily transmitted via the fecal-oral route: oocysts are excreted by infected individuals or animals (such as cattle), contaminating water and food sources, or transmitted through direct contact (such as caring for patients or changing diapers). Transmission can also occur through swimming pools.

[0003] Giardia lamblia has a tough cyst wall structure (mainly composed of chitin, protein, and carbohydrates). The key to nucleic acid lysis lies in effectively disrupting the cyst wall and the cell membrane of the parasite while avoiding nucleic acid degradation. The core challenges are: the cyst wall is tough and resistant to chemical / physical damage, and must be preferentially disrupted to release the parasite; the cell membrane contains lipids and proteins, which need further lysis to release nucleic acids; and preventing the degradation of DNA / RNA by nucleases (endogenous / exogenous) during lysis requires the simultaneous addition of inhibitors (such as EDTA and RNase inhibitors), placing high demands on the tolerance of the detection system.

[0004] Commonly used nucleic acid lysis methods are mainly physical lysis and chemical lysis. Physical lysis has disadvantages such as low lysis efficiency, easy RNA degradation (requiring strict temperature control), difficult sample processing, high instrument requirements, and is unsuitable for large-scale, mobile testing. Chemical lysis, while rapid lysis and avoidance of nucleic acid degradation, suffers from poor lysis results, cumbersome sample processing, and significant chemical reagent contamination, especially the high toxicity of guanidine salts, necessitating consideration of operator and environmental contamination. Furthermore, it can severely interfere with detection sensitivity and specificity.

[0005] Therefore, there is an urgent need for an extraction method that can efficiently and simultaneously break through the tough capsule walls of Giardia lamblia and Cryptosporidium, enabling rapid and stable release of nucleic acids, and is suitable for automation and large-scale water quality screening. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method for extracting nucleic acids from Giardia lamblia and Cryptosporidium and its application, which shortens the detection time, improves detection sensitivity and stability, and provides reliable technical support for drinking water safety monitoring.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for extracting nucleic acids from Giardia lamblia and / or Cryptosporidium, the method comprising: mixing a sample containing Giardia lamblia and / or Cryptosporidium with lysozyme and proteinase K, and incubating for digestion; adding lysis enhancement buffer and glass beads to the digested sample, performing sonication, centrifuging after sonication to obtain a supernatant; and performing automated nucleic acid extraction and purification on the obtained supernatant.

[0008] This invention employs a two-step pretreatment strategy of "enzymatic digestion-ultrasonic synergistic disruption," which shortens the sample processing time to within 3 hours, greatly improving detection efficiency, reducing detection costs, and providing excellent lysis results with high sensitivity and specificity.

[0009] Preferably, the method includes the following steps: (1) Sample pretreatment: Place the sample containing Giardia lamblia and / or Cryptosporidium into a centrifuge tube; (2) Enzyme digestion: Add lysozyme and proteinase K to the centrifuge tube from step (1), mix well, and incubate for digestion; (3) Ultrasonic disruption: Add lysis enhancement solution and glass beads to the digested sample in step (2), perform ultrasonic treatment, and centrifuge after ultrasonication to obtain supernatant; (4) Nucleic acid extraction: The supernatant obtained is subjected to automated nucleic acid extraction and purification using magnetic beads.

[0010] Preferably, the volume ratio of lysozyme and proteinase K in step (2) is 1 mL:(30-80) μL, for example 1 mL:30 μL, 1 mL:50 μL or 1 mL:80 μL.

[0011] Preferably, the incubation and digestion temperature in step (2) is 40-60°C (e.g., 40°C, 50°C or 60°C), and the incubation and digestion time is 55-75 min (e.g., 55 min, 60 min or 75 min).

[0012] Preferably, the ultrasound time in step (3) is 10-25 min (e.g., 10 min, 15 min or 20 min).

[0013] Preferably, the centrifugation speed in step (3) is 10,000-30,000 rpm (10,000 rpm, 20,000 rpm or 30,000 rpm), and the time is 2-5 min (e.g. 2 min, 3 min or 5 min).

[0014] Preferably, the automated nucleic acid extraction and purification of the obtained supernatant using the magnetic bead method in step (4) includes the following steps: (a) Combination: Mix the supernatant obtained by centrifugation with the magnetic beads for 1-2 min (e.g., 1 min or 2 min), and allow the magnetic beads to adhere to the magnetic beads for 30-90 min (e.g., 30 min, 60 min or 90 min). (b) Pyrolysis: Heating at 85-95℃ for 15-25 min (e.g., 15 min, 20 min or 25 min), followed by magnetic bead attraction for 100-150 min (e.g., 100 min, 120 min or 150 min). (c) Washing: After lysis, mix with washing solution 1 for 1-2 min (e.g., 1 min or 2 min), and magnetically attract the magnetic beads for 100-150 min (e.g., 100 min, 120 min or 150 min); then mix with washing solution 2 for 1-2 min (e.g., 1 min or 2 min), and magnetically attract the magnetic beads for 100-150 min (e.g., 100 min, 120 min or 150 min); then mix with washing solution 3 for 1-2 min (e.g., 1 min or 2 min), and magnetically attract the magnetic beads for 100-150 min (e.g., 100 min, 120 min or 150 min). (d) Elution: Elute with elution buffer at 80-90°C for 5-10 min (e.g., 5 min, 8 min or 10 min), air dry for 5-10 min (e.g., 5 min, 8 min or 10 min), magnetically attract with magnetic beads for 30-90 min (e.g., 30 min, 60 min or 90 min), discard the magnetic beads, and obtain a solution containing Giardia lamblia and / or Cryptosporidium nucleic acid.

[0015] Preferably, the washing liquid 1 comprises any one or a combination of at least two of guanidine hydrochloride, guanidine isothiocyanate, or a descaling agent.

[0016] Preferably, the washing liquid 2 comprises any one or a combination of at least two of sodium chloride solution, ethanol-isopropanol or Tris.

[0017] Preferably, the washing liquid 3 includes acetone.

[0018] Preferably, the eluent comprises EDTA and / or Tris.

[0019] As a preferred technical solution, the present invention provides a method for extracting nucleic acids from Giardia lamblia and / or Cryptosporidium, as follows: (1) Sample pretreatment: Cut the filter paper enriched with Giardia lamblia and / or Cryptosporidium into small pieces and place them in a centrifuge tube; (2) Enzyme digestion: Add lysozyme and proteinase K in a volume ratio of 1 mL:(30-80) μL to the centrifuge tube of step (1), mix well, and incubate at 40-60℃ for 55-75 min. (3) Ultrasonic disruption: Add lysis enhancement solution and glass beads to the sample digested in step (2), and perform ultrasonic treatment. After ultrasonic treatment for 10-25 min, centrifuge at 10000-30000 rpm for 2-5 min to obtain supernatant. (4) Nucleic acid extraction: Mix the supernatant obtained by centrifugation with magnetic beads for 1-2 min, and magnetic beads adsorb for 30-90 min; heat and lyse at 85-95℃ for 15-25 min, and magnetic beads adsorb for 100-150 min; after lysis, mix with washing solution 1 for 1-2 min, and magnetic beads adsorb for 100-150 min; then mix with washing solution 2 for 1-2 min, and magnetic beads adsorb for 100-150 min; then mix with washing solution 3 for 1-2 min, and magnetic beads adsorb for 100-150 min; wash with elution solution at 80-90℃ for 5-10 min, air dry for 5-10 min, and magnetic beads adsorb for 30-90 min; discard the magnetic beads to obtain a solution containing Giardia lamblia and / or Cryptosporidium nucleic acid.

[0020] Secondly, the present invention provides the application of the method described in the first aspect in the preparation of products for detecting Giardia lamblia and / or Cryptosporidium nucleic acids in tap water.

[0021] Thirdly, the present invention provides the application of the method described in the first aspect in the safety monitoring of tap water quality.

[0022] Fourthly, the present invention provides the use of the method described in the first aspect in the preparation of in vitro diagnostic reagents for the diagnosis of Giardia lamblia and / or Cryptosporidium infection.

[0023] Fifthly, the present invention provides a method for detecting Giardia lamblia and / or Cryptosporidium nucleic acid in tap water, the method comprising: extracting Giardia lamblia and / or Cryptosporidium nucleic acid using the method described in the first aspect, and amplifying and detecting the extracted nucleic acid using isothermal amplification technology.

[0024] In one embodiment of the present invention, the nucleic acid sequence of the primers used for isothermal amplification includes the sequences shown in SEQ ID NO.1-SEQ ID NO.4, and the nucleic acid sequence of the probe includes the sequences shown in SEQ ID NO.5-6.

[0025] Giardia lamblia forward primer (SEQ ID NO.1): CTCAGGAAGGAGGCCCTCAAGAGCCTGAACGA.

[0026] Giardia lamblia reverse primer (SEQ ID NO.2): TCTCGCGGGCGATCGTCTCCTTCTCGATSGC.

[0027] Probe (SEQ ID NO.5): TYGCCACGGAGAACGCMGARAGGAAGAAGA / i6FAMdT / / idSp / / iBHQ1dT / AYGACCAGCTCAACG.

[0028] Cryptosporidium forward primer (SEQ ID NO.3): TGAGAAACGGCTACCACATCTAAGGAAGGC.

[0029] Cryptosporidium reverse primer (SEQ ID NO.4): CTCCAATTGATACTYGTWAAGGGGTTTATAC.

[0030] Probe (SEQ ID NO.6): CGCAAATTACCCAATCCTRAYACAGGGAGG / i6FAMdT / A / idSp / / iBHQ1dT / GACAAGAAATAACAAT.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) High lysis efficiency and good extraction synchronization: The synergistic strategy of "enzyme digestion + ultrasound" specifically breaks down the complex capsule wall of the two insects, solves the problem of incomplete lysis by a single method, and realizes efficient and synchronous release of nucleic acids from the two insects. (2) Simplified process, fast and efficient: "Extraction with filter paper" is adopted, eliminating the elution step; the optimal parameter combination shortens the key pretreatment time to 75 min (digestion 60 min + sonication 15 min), and combined with automated magnetic bead extraction, the whole process can be completed within 3 hours, which greatly improves the detection efficiency; (3) High sensitivity and good repeatability: The method of the present invention can still effectively detect samples with as few as 10 Cryptosporidium oocysts / tubes, and has excellent intra- and inter-batch repeatability, which significantly reduces the risk of false negatives; (4) Safe and environmentally friendly, easy to promote: It avoids the use of highly toxic chemical lysis agents and mainly adopts biological enzymes and physical methods. It is safe, the method is compatible with conventional laboratory instruments and automated extraction platforms, and it is easy to standardize and apply on a large scale. (5) Broad application prospects: The method of the present invention is not only applicable to tap water monitoring, but can also be extended to the detection of "two parasites" in various water bodies such as surface water, drinking water plant process water, and swimming pool water, as well as in clinical and food safety related fields. Attached Figure Description

[0032] Figure 1 The results of detection after 60 min of digestion with lysozyme / proteinase K followed by 15 min of sonication are shown in the figure. Figure 2 The image shows the detection results after lysozyme / proteinase K digestion for 30 min followed by sonication for 15 min. Figure 3 The results of detection after 60 min of digestion with lysozyme / proteinase K followed by 30 min of sonication are shown in the figure. Figure 4 The image shows the detection results after lysozyme / proteinase K digestion for 30 min followed by sonication for 30 min. Figure 5 The image shows the test results for 50 oocysts / tubes of the standard sample; Figure 6 The image shows the test results of 25 oocysts / tubes of the standard sample; Figure 7 The image shows the test results of 10 oocytes / tubes for the standard sample. Detailed Implementation

[0033] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] In the following examples, washing solution 1 is guanidine isothiocyanate (purchased from Sinopharm Chemical Reagent Co., Ltd., model: 593-84-0); washing solution 2 is isopropanol (purchased from Sinopharm Chemical Reagent Co., Ltd., model: 67-63-0); and washing solution 3 is a self-prepared alcohol-free washing solution used to remove impurities.

[0036] Example 1 Verification of Giardia lamblia extraction results.

[0037] (1) Take two samples of Giardia lamblia standard: 15,000 cysts / tube and 7,500 cysts / tube, and take one sample of negative water as the negative control group (NC). Add 2 L of tap water to each sample and mix thoroughly. (2) After the above samples were filtered and enriched by the two parasites-specific filtration enrichment device jointly developed by Jiangsu Qitian Gene Biotechnology Co., Ltd. and Jiangsu Institute of Schistosomiasis Control (see CN216236324U and CN218860716U), the filter paper enriched with tap water was cut into pieces with sterile scissors and placed in 2 mL centrifuge tubes respectively. (3) Add 0.8 mL of lysozyme and 40 μL of proteinase K to the centrifuge tubes, shake well and incubate at 50°C for 60 min for digestion; (4) Add 50 μL of MP-A solution and 100 μL of glass beads to the above sample tubes respectively, and then place them in an ultrasonic homogenizer for ultrasonic treatment for 15 min; (5) Place the ultrasonically treated sample tube in a high-speed centrifuge and centrifuge at 13,000 rpm for 2 min, then take the supernatant. (6) Take the solution from the 96-well plate: dispense 500 μL of lysis buffer into column 1 / 7, dispense 400 μL of the above suspension into column 2 / 8, dispense 700 μL of washing buffer 1 into column 3 / 9, dispense 700 μL of washing buffer 2 into column 4 / 10, dispense 700 μL of washing buffer 3 into column 5 / 11, and dispense 100 μL of elution buffer into column 6 / 12; (7) Move the magnetic beads into column 2 / 8, mix for 1 min, and magnetic attraction time for 60 min; (8) Transfer the solution from the above steps into column 1 / 7 and heat it for pyrolysis. The heating temperature is 90℃, the mixing and pyrolysis time is 20 min, and the magnetic bead adsorption time is 120 min. (9) Transfer the above solution to column 3 / 9 for washing, mix for 2 min, and magnetic bead adsorption time for 120 min; (10) Transfer the above solution to column 4 / 10 for washing, mix for 2 min, and magnetic bead adsorption time for 120 min; (11) Transfer the above solution to column 5 / 11 for washing, mix for 1 min, and magnetic bead adsorption time for 120 min; (12) Transfer the above solution to column 6 / 12 for elution, heat to 85°C, elute and mix for 5 min, and air dry for 5 min. Magnetic bead adsorption time is 60 min. (13) Transfer the above solution to a clean 1.5 mL centrifuge tube, centrifuge at high speed (13000 rpm) to remove the magnetic beads, take 50 μL of the supernatant and store it at -20℃ for later use; (14) Detection procedure: Prepare a 50 μL reaction system: 25 μL reaction buffer, 12.7 μL double-distilled water, 2.1 μL forward primer SEQ ID NO.1 (420 nmol / L), 2.1 μL reverse primer SEQ ID NO.2 (420 nmol / L), and 0.6 μL probe SEQ ID NO.3 (120 nmol / L). After mixing, add to the RAA reaction unit lyophilized powder (model: RT-RAA fluorescence method) produced by Qitian Gene Company. After reconstitution, mix by hand, centrifuge briefly, add 2.5 μL of magnesium acetate solution with a concentration of 280 mmol / L to the inner wall of the reaction unit tube cap, and then add 5 μL of DNA template to the reaction unit. Place it in the sample pretreatment system (model: B6108) and mix thoroughly (temperature 39℃) for 15 min. Take it out and place it in the isothermal nucleic acid amplification analyzer (model F1620 / F1628) for detection. Record the analysis results. The results are as follows. Figure 1 As shown, both concentration standards yielded stable and specific positive amplification curves, indicating that longer enzyme digestion and sonication time are not necessarily better; there exists a precise optimal combination.

[0038] Example 2 The difference between this example and Example 1 is that the lysozyme / proteinase K digestion time is 75 min and the sonication time is 10 min. As a result, both concentration standards gave stable and specific positive amplification curves.

[0039] Example 3 The difference between this example and Example 1 is that the lysozyme / proteinase K digestion time is 55 min and the sonication time is 25 min. As a result, both concentration standards gave stable and specific positive amplification curves.

[0040] Example 4 The difference between this embodiment and Example 1 is that the lysozyme / proteinase K digestion time is 30 min, and the results are as follows: Figure 2 As shown, no detection was found in 7500 samples per tube, indicating unstable results.

[0041] Example 5 The difference between this embodiment and Embodiment 1 is that the ultrasound time is 30 minutes, and the results are as follows: Figure 3 As shown, 7500 samples per tube were not completely detected, indicating that excessive ultrasound caused severe damage to nucleic acids and disrupted the nucleic acid release mechanism.

[0042] Example 6 The difference between this embodiment and Example 1 is that the lysozyme / proteinase K digestion time is 30 min and the sonication time is 30 min. The results are as follows: Figure 4 As shown, no peaks were observed in any of the standards, and no samples of any concentration were detected; all test results were negative.

[0043] Example 7 Sensitivity verification of Cryptosporidium extraction.

[0044] Following the extraction method described in Example 1, Cryptosporidium oocyst standards (50, 25, and 10 oocysts / tube) were processed and nucleic acid extracted, followed by isothermal amplification and detection.

[0045] Result: As Figures 5-7 As shown, Cryptosporidium samples at all three concentration gradients were successfully detected, exhibiting typical positive amplification curves, while the negative control showed no amplification. This indicates that the method of the present invention has high extraction efficiency and detection sensitivity for low-load Cryptosporidium oocysts.

[0046] Example 8 Full-process time and repeatability verification.

[0047] To simulate a real-world tap water monitoring scenario, ten 2-liter tap water samples containing the two parasites ("two parasites") were collected. Starting with filter paper enrichment, the method described in Example 1 of this invention (75-minute pretreatment and automated magnetic bead extraction) was fully executed, followed by isothermal amplification detection. Simultaneously, parallel samples were treated using the traditional guanidine salt-ultrasonic method as controls.

[0048] Results: The method of this invention takes an average of approximately 2.8 hours for the entire process, achieves a 100% detection rate for 10 samples, and has a coefficient of variation of less than 5% for the threshold time (Tt value) of the amplification curve. Traditional methods take more than 6 hours, have a detection rate of only 80%, and exhibit significant variation in Tt values. This demonstrates the significant advantages of this invention in terms of speed and repeatability.

[0049] In summary, this invention employs a two-step pretreatment strategy of "enzymatic digestion-ultrasonic synergistic disruption" to integrate two pathogens into one tube for simultaneous extraction and detection. This reduces sample processing time to within 3 hours, significantly improving detection efficiency, lowering detection costs, and demonstrating excellent lysis results with high sensitivity and specificity.

[0050] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for extracting nucleic acids from Giardia lamblia and / or Cryptosporidium, characterized in that, The method includes: mixing a sample containing Giardia lamblia and / or Cryptosporidium with lysozyme and proteinase K, and incubating for digestion; adding lysis enhancement solution and glass beads to the digested sample, performing sonication, centrifuging after sonication to obtain supernatant; and performing automated nucleic acid extraction and purification on the obtained supernatant.

2. The method according to claim 1, characterized in that, The method includes the following steps: (1) Sample pretreatment: Place the sample containing Giardia lamblia and / or Cryptosporidium into a centrifuge tube; (2) Enzyme digestion: Add lysozyme and proteinase K to the centrifuge tube from step (1), mix well, and incubate for digestion; (3) Ultrasonic disruption: Add lysis enhancement solution and glass beads to the digested sample in step (2), perform ultrasonic treatment, and centrifuge after ultrasonication to obtain supernatant; (4) Nucleic acid extraction: The supernatant obtained is subjected to automated nucleic acid extraction and purification using magnetic beads.

3. The method according to claim 2, characterized in that, The volume ratio of lysozyme and proteinase K in step (2) is 1 mL:(30-80) μL; Preferably, the incubation and digestion temperature in step (2) is 40-60°C, and the incubation and digestion time is 55-75 min.

4. The method according to claim 2 or 3, characterized in that, The ultrasound time in step (3) is 10-25 min.

5. The method according to any one of claims 2-4, characterized in that, The centrifugation speed in step (3) is 10,000-30,000 rpm, and the time is 2-5 min.

6. The method according to any one of claims 2-5, characterized in that, The automated nucleic acid extraction and purification of the obtained supernatant using the magnetic bead method described in step (4) includes the following steps: (a) Combination: Mix the supernatant obtained by centrifugation with the magnetic beads for 1-2 min, and let the magnetic beads magnetically attract for 30-90 min; (b) Pyrolysis: Heating at 85-95℃ for 15-25 min, followed by magnetic bead attraction for 100-150 min; (c) Washing: After pyrolysis, mix with washing solution 1 for 1-2 min, and magnetic beads are attracted for 100-150 min; then mix with washing solution 2 for 1-2 min, and magnetic beads are attracted for 100-150 min; then mix with washing solution 3 for 1-2 min, and magnetic beads are attracted for 100-150 min. (d) Elution: Elute with elution buffer at 80-90℃ for 5-10 min, air dry for 5-10 min, magnetically attract with magnetic beads for 30-90 min, discard the magnetic beads, and obtain a solution containing Giardia lamblia 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 descaling agent; 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 includes acetone; Preferably, the eluent comprises EDTA and / or Tris.

7. The use of the method according to any one of claims 1-6 in the preparation of a product for detecting Giardia lamblia and / or Cryptosporidium nucleic acid in tap water.

8. The application of the method according to any one of claims 1-6 in the safety monitoring of tap water quality.

9. The use of the method of any one of claims 1-6 in the preparation of in vitro diagnostic reagents for the diagnosis of Giardia lamblia and / or Cryptosporidium infection.

10. A method for detecting Giardia lamblia and / or Cryptosporidium nucleic acid in tap water, characterized in that, The method includes: extracting Giardia lamblia and / or Cryptosporidium nucleic acid using the method described in any one of claims 1-6, and amplifying and detecting the extracted nucleic acid using isothermal amplification technology.

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