CN108950063A - For detecting the kit and method that VZV infects in eye micro-biological sample - Google Patents

Abstract

The present invention "kit and method for detecting VZV infection in microscopic biological samples of the eye" relates to molecular detection technology of ocular virus infection, especially a method for extracting DNA from microbiological samples, the microbiological sample refers to the volume Liquid samples not exceeding 10ul‑200ul or solid samples not exceeding 1×1mm in volume. The extracted DNA is used as a template for PCR detection, which can detect eye virus infection with an accuracy of over 80%, providing a reliable basis for subsequent appropriate treatment methods and reducing the blindness of treatment methods.

Description

Kits and methods for detecting VZV infection in ocular trace biological samples

technical field

The invention relates to a molecular detection technology of virus infection, in particular to a kit and a method for detecting VZV infection in eye trace biological samples.

technical background

Due to the complex structure of the eye and the influence of the blood-brain barrier, the correlation between many detection indicators of eye tissue samples and blood detection indicators is very low. A large number of clinical data studies have found that blood tests cannot accurately reflect the true cause of the eye, especially The etiology of infectious eye diseases; on the other hand, eye specimens are small in size and have a variety of specimen types, including various tissues, various body fluids (vitreous, aqueous humor, tears, etc.), various membranes (retina, corneal endothelium, etc.) ), multi-site secretions, etc., the viral DNA content in ocular liquid samples is extremely low, while the viral DNA in solid samples is difficult to extract, and the sample content is extremely complex, and is limited by the extremely small sampling volume (of which solid samples are generally No more than 1mm ³ ; in addition to tears, the amount of eye fluid specimens that can generally be taken is about 20-100ul, and the maximum is no more than 200ul) It is difficult to meet the large volume and uniform type required by blood and urine tests, so eye specimens are used Conventional blood testing methods have a very high rate of false negatives and often delay treatment. At present, there is no virus detection technology suitable for micro-specimens, especially micro-specimens taken from the eyes.

Varicella-Zoster Virus (VZV) is morphologically identical to VZV. There is only one serotype. There are 71 genes in the genome, encoding 67 different proteins, including 6 glycoproteins, which are now collectively named gE, gB, gH, gI, gC and gL. Glycoproteins gE, gB and gH are extremely abundant in infected cells and are also present in the membrane of virions. VZV has no animal storage host, and humans are the only natural host. There are two types of VZV infection, namely primary infection with chickenpox (varicella) and recurrent infection with herpes zoster (zoster). The skin is the main target organ of the virus. In patients who have suffered from chickenpox, a small amount of virus is latent in the sensory ganglia of the dorsal root ganglia of the spinal cord or cranial nerves. The latent virus can be reactivated to cause herpes zoster. When the immunity is weakened due to some other diseases such as trauma, long-term sub-health, surgery, etc., or the use of immunosuppressive drugs, the virus can be revived and the activated The virus descends through the axons of sensory nerve fibers to the innervated skin area, and causes herpes zoster after multiplying. In the early stage, the local skin feels abnormal, itchy and painful, and then rashes and herpes appear, which are connected in series and form bands, which are more common on the trunk and face, and are distributed unilaterally. The course of the disease is about 3 weeks, and a few can last for several months. Long.

Herpes zoster virus infection is a clinically common and difficult to tolerate disease, which is caused by the semilunar ganglion or a branch of the trigeminal nerve being infected by varicella zoster virus. It has the following clinical characteristics: (1) skin lesions are the most common, and the characteristics of skin lesions are bright blisters the size of mung beans clustered on inflammatory erythema, and the blister fluid is absorbed, dried up, and scabbed afterwards. Each group of skin lesions is separated by normal skin, arranged in a band, and the lesions are strictly limited to the innervation area but not the midline of the face. If the blisters are combined with bacterial infection, the redness, swelling and pain in the lesion area will be aggravated, and eventually suppurate. Scars may be left after healing, and even the hair on the damaged area cannot be regenerated. All cases in this group had skin lesions. If they were diagnosed and treated in time, the lesions were mild. If they were not diagnosed and treated in time or combined with bacterial infection, the skin lesions could be quite serious. (2) Acute retinal necrosis (ARN), 80% is caused by VZV infection. Causes irreversible damage to the retina and is highly blinding. (3) Corneal lesions are relatively common, accounting for 50% of the cases in this group. Most of them are punctate corneal epithelial erosions, often accompanied by insensitivity, which can cause pseudodendritic keratitis. The lesions are located in the peripheral part of the cornea and manifest as dendritic spots. The corneal stroma is diffusely edematous, the fluorescein is not stained, and it responds well to steroid therapy. Sometimes it is easily misdiagnosed as true dendritic keratitis caused by herpes simplex. Dendritic keratitis vera is often located in the center of the cornea, with thin branches and enlarged ends, epithelial defect, fluorescein staining, and worsening by steroid treatment. Early herpes zoster lesions on the upper end of the nose or near the nose innervated by the nasociliary nerve indicate the harbinger of keratitis, but some people think that it cannot be predicted because of the involvement of the nasociliary nerve. Keratitis occurs in all patients with nasal ciliary nerve involvement, but those with keratitis do not necessarily have nasal ciliary nerve involvement. (4) The incidence rate of iridocyclitis is 15%, most of them are mild and moderate iridocyclitis, KP and Tyndale's sign are positive. (5) The incidence rate of secondary glaucoma in this group of patients was 12%. All had severe iridocyclitis. It is generally believed that increased intraocular pressure can be caused by pigment granules in the aqueous humor, cell debris blocking the trabecula, or trabecular inflammation and anterior synechia around the iris. (6) Ophthalmic neurological complications caused by herpes zoster of the eye are more commonly involving the third cranial nerve.

At present, blood tests are still used to detect ocular viral infections. However, a large number of clinical data studies have found that blood tests cannot accurately reflect the real cause of the eye, especially the cause of infectious eye diseases, which is easily missed and misdiagnosed.

Obtaining accurate detection results of eye infection is the prerequisite for correct treatment in the future; however, there is still a lack of highly sensitive detection methods specifically for virus infection in ocular tissues, and there is no conventional method using micro-sample of the eye as a specimen. PCR detection of viral infection with high sensitivity has been reported.

Contents of the invention

According to the gaps in the above fields, it is urgent to develop a virus DNA extraction method and a high-sensitivity virus detection kit suitable for small and diverse ocular specimens clinically. The method for virus detection, the fluorescent probe detection kit and the DNA extraction method, the technical scheme claimed by the present invention is as follows:

A method for preparing a DNA template of a trace biological sample, comprising the steps of:

(1) Put the micro-biological sample to be tested into a container, add 10-30 μl proteinase K, 100-300 μl lysis buffer AL, and treat at 56°C for at least 10 minutes; the micro-biological sample refers to a volume of no more than 10ul-200ul Liquid samples or solid samples whose volume does not exceed ^1mm3 ;

(2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

(3) Put the liquid obtained in step (2) into a spin column, put it into a 2ml collection tube, and centrifuge at 6000-9000rpm for 0.5-2min;

If the sample volume > 140 μl, repeat the above step (3);

(4) Add 300-600μl elution buffer 1, 6000-9000rpm, 0.5-2min, discard the filtrate and collection tube, and replace with a new collection tube;

(5) Add 300-600μl Elution Buffer 2, centrifuge at 10000-15000rpm for 1-5min, discard the filtrate and collection tube;

(6) Replace with a new 1.5ml liquid centrifuge tube, centrifuge at 10000-15000rpm, centrifuge for 0.5-2min, discard the filtrate and centrifuge tube;

(7) Replace with a new 1.5ml centrifuge tube, add 50-100 μl of elution buffer 3, place at room temperature for 0.5-2min, centrifuge at 6000-9000rpm for 0.5-2min to obtain the DNA of the trace biological sample to be tested;

in:

The lysis buffer is Tris saturated phenol containing 10% SDS;

The formula of described elution buffer 1 is: saturated phenol: chloroform: the mixed solution of isoamyl alcohol with volume ratio 25:24:1;

The elution buffer 2 is absolute ethanol;

The elution buffer 3 is pH 8.0, 10 mmol/L Tris-HCl solution containing 1 mmol/L EDTA.

Preferably, the steps are as follows:

(1) Put the trace biological sample to be tested into a container, add 20 μl proteinase K, 200 μl lysis buffer, and treat at 56°C for at least 10 minutes;

(2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

(3) Put the liquid obtained in step (2) into a spin column, put it into a 2ml collection tube, and centrifuge at 6000-9000rpm for 0.5-2min; if the sample volume is > 140μl, repeat the above step (3);

(4) Add 500 μl of elution buffer at 1,8000 rpm, centrifuge for 1 min, discard the filtrate and collection tube, and replace with a new collection tube;

(5) Add 500 μl of elution buffer 2, centrifuge at 14000 rpm for 3 minutes, discard the filtrate and collection tube;

(6) Replace with a new 1.5ml centrifuge tube, centrifuge at 14000rpm, centrifuge for 1min, and discard the filtrate and centrifuge tube;

(7) Change to a new 1.5ml centrifuge tube, add 50-100 μl of elution buffer 3, place at room temperature for 1 min, and centrifuge at 8000 rpm for 1 min to obtain the DNA of the trace biological sample to be tested.

Preferably, the micrometric biological sample is a liquid sample, and after being treated at 56°C for 10 minutes in step (1), it is then placed in an environment of 70°C for 10 minutes.

Preferably, the micrometric biological sample is a solid tissue, which is treated at 56° C. for 5-12 hours in step (1).

Preferably, the liquid sample refers to ocular secretion, aqueous humor, tear fluid and/or vitreous body; the solid tissue refers to retina, corneal endothelium, pterygium, conjunctiva, iris and/or ocular tumor.

The present invention also provides a reagent combination for preparing DNA templates of trace biological samples, which is characterized in that it comprises the following reagents;

Lysis buffer: Tris-saturated phenol with 10% SDS,

Elution buffer 1: a mixture of saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1;

Elution buffer 2: absolute ethanol,

Elution buffer 3: pH8.0, 10mmol/L Tris-HCl solution containing 1mmol/LEDTA,

The eye trace sample refers to a liquid sample whose volume does not exceed 10ul-200ul or a solid sample whose volume does not exceed ^1mm3 ;

The liquid sample refers to ocular secretions, aqueous humor, tears and/or vitreous;

The solid tissue refers to retina, corneal endothelium, pterygium, conjunctiva, iris and/or ocular tumors.

The present invention further provides a PCR amplification reagent for detecting VZV infection in eye trace samples, characterized in that:

Contains the following primer probe set, the sequence is as follows:

Primer-F: AACACCCGACTCGAAATACCA

Primer-R: TATTGGCACGCAACTCAACTG

Probe: AACTCAACTGGCACGCTTCG

The eye trace sample refers to a liquid sample whose volume does not exceed 10ul-200ul or a solid sample whose volume does not exceed ^1mm3 ;

The liquid sample refers to ocular secretions, aqueous humor, tears and/or vitreous;

The solid tissue refers to retina, corneal endothelium, pterygium, conjunctiva, iris and/or ocular tumors.

Further, the PCR amplification system used for each reaction contains:

The PCR amplification reagent is added with eye micro-sample DNA template and distilled water before use to make the total reaction volume reach 20 volume units.

Preferably, the volume unit may be 1 microliter, 1.5 microliter, 2 microliter or 2.5 microliter.

Further, the PCR amplification system used for each reaction contains:

In another aspect, the present invention also provides a kit for detecting ocular VZV infection through eye micro-sample, characterized in that: comprising

Any of the above-mentioned amplification reagents for detecting VZV infection in micro-sample of the eye,

As well as the above-mentioned reagent set for preparing DNA templates for micro-sample of eyes,

Wherein, the eye micro-sample refers to a liquid sample whose volume does not exceed 10ul-200ul or a solid sample whose volume does not exceed 1×1mm.

Preferably, the kit also includes a control template for setting a control reaction for each detection reaction, specifically as follows:

Positive control template: VZV virus fragment;

Positive control template: positive ocular body fluid;

Blank control template: ultrapure water;

Negative control template: Negative eye fluid sample;

Weak positive control template: the diluent of positive ocular body fluid, and its critical value is set at ΔT=38-40.

Another aspect of the present invention provides a PCR method for detecting ocular VZV infection through eye micro-sample, characterized in that,

It includes preparing the eye trace sample DNA template, and then using any of the PCR amplification reagents to perform real-time quantitative PCR detection on the eye trace sample DNA template;

The steps for preparing the eye micro sample DNA template are as follows:

(1) Put the micro-biological sample to be tested into a container, add 10-30 μl proteinase K, 100-300 μl lysis buffer AL, and treat at 56°C for at least 10 minutes; the micro-biological sample refers to a volume of no more than 10ul-200ul Liquid samples or solid samples whose volume does not exceed 1×1mm;

(2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

(3) Put the liquid obtained in step (2) into a spin column, put it into a 2ml collection tube, and centrifuge at 6000-9000rpm for 0.5-2min;

If the sample volume > 140 μl, repeat the above step (3);

(4) Add 300-600μl elution buffer 1, 6000-9000rpm, 0.5-2min, discard the filtrate and collection tube, and replace with a new collection tube;

(5) Add 300-600μl Elution Buffer 2, centrifuge at 10000-15000rpm for 1-5min, discard the filtrate and collection tube;

(6) Replace with a new 1.5ml liquid centrifuge tube, centrifuge at 10000-15000rpm, centrifuge for 0.5-2min, discard the filtrate and centrifuge tube;

(7) Replace with a new 1.5ml centrifuge tube, add 50-100 μl of elution buffer 3, place at room temperature for 0.5-2min, centrifuge at 6000-9000rpm for 0.5-2min to obtain the DNA template of the trace biological sample to be tested.

in:

The lysis buffer is Tris saturated phenol containing 10% SDS;

The formula of described elution buffer 1 is: saturated phenol: chloroform: the mixed solution of isoamyl alcohol with volume ratio 25:24:1;

The elution buffer 2 is absolute ethanol;

The elution buffer 3 is pH8.0, containing 10mmol/L Tris-HCl solution of 1mmol/L EDTA;

The eye micro-sample refers to a liquid sample with a volume of no more than 10ul ^- 200ul or a solid sample with a volume of no more than 1mm; the liquid sample refers to ocular secretions, aqueous humor, tears and/or vitreous; the solid tissue Refers to retinal, corneal endothelium, pterygium, conjunctival, iris, and/or ocular tumors.

Preferably, the steps of preparing the eye micro sample DNA template are as follows:

(1) Put the trace biological sample to be tested into a container, add 20 μl proteinase K, 200 μl lysis buffer, and treat at 56°C for at least 10 minutes;

(2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

(3) Put the liquid obtained in step (2) into a spin column, put it into a 2ml collection tube, and centrifuge at 6000-9000rpm for 0.5-2min; if the sample volume is > 140μl, repeat the above step (3);

(4) Add 500 μl of elution buffer at 1,8000 rpm, centrifuge for 1 min, discard the filtrate and collection tube, and replace with a new collection tube;

(5) Add 500 μl of elution buffer 2, centrifuge at 14000 rpm for 3 minutes, discard the filtrate and collection tube;

(6) Replace with a new 1.5ml centrifuge tube, centrifuge at 14000rpm, centrifuge for 1min, and discard the filtrate and centrifuge tube;

(7) Replace with a new 1.5ml centrifuge tube, add 50-100 μl of elution buffer 3, place at room temperature for 1 min, centrifuge at 8000 rpm for 1 min to obtain the DNA of the trace biological sample to be tested.

Preferably, when the micrometric biological sample is a liquid, after being treated at 56° C. for 10 minutes in step (1), it is then placed in an environment of 70° C. for 10 minutes.

Preferably, when the micrometric biological sample is a solid tissue, it is treated at 56° C. for 5-12 hours in step (1).

Preferably, the program of the PCR reaction is as follows:

37℃×2min

94℃×2min, the above two temperature and time cycle 40 times

93℃×15sec,

Fluorescence was detected at 60°C.

One of the main contributions of the present invention is that it proposes to extract DNA from a micro sample taken from the eye to prepare a PCR template, and to use real-time quantitative PCR (real time-PCR) to detect eye virus infection.

Due to the complex structure of the eye and the influence of the blood-brain barrier, the correlation between many detection indicators of eye tissue samples and blood detection indicators is very low. A large number of clinical data studies have found that blood tests cannot accurately reflect the true cause of the eye, especially The etiology of infectious eye diseases; on the other hand, eye specimens are small in size and have a variety of specimen types, including various tissues, various body fluids (vitreous, aqueous humor, tears, etc.), various membranes (retina, corneal endothelium, etc.) ), multi-site secretions, etc., in which solid specimens generally do not exceed 1mm ³ ; except for tears, the amount of eye liquid specimens that can generally be taken is about 20-100ul, and the maximum is no more than 200ul, which is difficult to meet the requirements of blood and urine tests. Because of the large volume and homogeneous species, routine blood testing methods for ocular specimens have a very high rate of false negatives, often delaying treatment. At present, there is no virus detection technology suitable for micro-specimens, especially micro-specimens taken from the eyes.

Based on this, the inventor explored a DNA extraction method suitable for micro-tissue samples, and explored the volume, time, temperature, and dosage of extraction reagents such as proteinase K, lysate, and elution buffer, and proposed a simple and easy-to-operate method. A method for extracting micro-tissue DNA, which is suitable for extracting DNA templates of various ocular clinical specimens, and the obtained templates are used for PCR detection of the present invention, which can detect VZV virus infection in ocular micro-tissues with high sensitivity.

For example, the DNA in 10ul of tear fluid is enough to serve as a template for 4 PCR reactions, and the virus infection in the eye can be detected successfully through tear fluid detection. This method, this DNA extraction method, is not only suitable for a variety of microscopic specimens of the eye, but also for other biological specimens that are difficult to obtain, such as tissue samples from infants and tissue samples from tiny organisms, and is non-invasive or minimally traumatic to patients change.

Another main contribution of the present invention is that it also provides a kit for detecting virus infection, especially VZV infection, and provides a PCR amplification reagent for detecting VZV, a primer probe set, combined with the above DNA extraction method, to carry out detection. Experimental data show that whether it is clinical ocular micro-samples collected at that time or various ocular micro-samples sent from other urban hospitals or hospitals for more than 24-48 hours, it can detect VZV infection stably and sensitively. The method Has good stability and reliability.

In view of the small amount and preciousness of eye samples, in order to reduce the misjudgment of results and sample waste caused by human error, reagent contamination, failure and other reasons during the detection process. In the preferred embodiment of the kit of the present invention, multiple control templates are set for each detection reaction, wherein:

In view of the small amount and preciousness of eye samples, in order to reduce the misjudgment of results and sample waste caused by human error, reagent contamination, failure and other reasons during the detection process. In the preferred embodiment of the kit of the present invention, multiple control templates are set for each detection reaction, wherein:

Positive control template: known virus VZV DNA; the role is to control the effectiveness of the detection system, reagents and equipment to prevent false negative results

Positive control template: Positive ocular body fluid; used to indicate the effectiveness of the ocular specimen detection system, thereby eliminating false negatives.

Blank control template: ultrapure water; used to indicate whether there is contamination in the reaction reagent, so as to indicate whether there is a false positive in the result at the same time.

Negative control template: Negative eye fluid sample; used to show the normal noise of eye samples and indicate whether there are false positives caused by contamination.

Weak positive control template: Due to the small size of eye specimens and low viral DNA content, a weak positive control is required to determine the sensitivity of the detection.

Another main contribution of the present invention is to provide a PCR method for detecting VZV infection in trace tissue samples, which can accurately detect VZV infection in trace samples by adjusting the PCR system and procedures.

Description of drawings

Figure 1. Intraocular viral endotheliitis

Fig. 2. After the eyeball shown in Fig. 1 was confirmed by virus detection, ganciclovir eye gel and acyclovir oral treatment were used for one week, and the vision improved obviously.

Figure 3 is a schematic diagram of the results of PCR detection of VZV infection in one of the samples to be tested.

Detailed ways

The technical solutions of the present invention are exemplified below through specific examples, but are not intended to limit the patent scope of the present invention.

Lysis buffer: Tris-saturated phenol with 10% SDS,

Elution buffer 1: a mixture of saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1;

Elution buffer 2: absolute ethanol,

Elution buffer 3: pH 8.0, 10 mmol/L Tris-HCl solution containing 1 mmol/LEDTA.

Control nucleic acid extraction kit: purchased from Daan Gene Co. , Ltd.

Control method: VZV detection kit: purchased from Daan Gene Co. , Ltd.

Example 1. Method for extracting DNA from test specimens of eye microfluidics

Material:

Specimens to be tested: collected from admitted patients, tear fluid, aqueous humor 2, and vitreous.

(1) Put the collected sample to be tested into a container, add 10-30 μl proteinase K, 100-300 μl lysis buffer AL, and treat at 56°C for more than 10 minutes;

(2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

(3) Put the liquid obtained in step (2) into a spin column, put it into a 2ml collection tube, and centrifuge at 6000-9000rpm for 0.5-2min;

If the sample volume > 140 μl, repeat step (3);

(4) Add 300-600μl elution buffer 1, 6000-9000rpm, 0.5-2min, discard the filtrate and collection tube, and replace with a new collection tube;

(5) Add 300-600μl Elution Buffer 2, centrifuge at 10000-15000rpm for 1-5min, discard the filtrate and collection tube;

(6) Replace with a new 1.5ml liquid centrifuge tube, centrifuge at 10000-15000rpm, centrifuge for 0.5-2min, discard the filtrate and centrifuge tube;

(7) Change to a new 1.5ml centrifuge tube, add 50-100μl elution buffer 3, and place at room temperature for 0.5-2min,

Centrifuge at 6000-9000rpm for 0.5-2min to obtain the DNA template of the trace biological sample to be tested, and finally 50-100ul DNA template can be obtained.

(8) Take 1ul sample, detect OD260 and OD280 by DNA content detection instrument nanodrop2000, and calculate the DNA concentration to be 30-70ng/μl)

Control: Use a nucleic acid extraction kit (purchased from Daan Gene . As a control reagent, operate DNA extraction and PCR amplification according to its instructions.

Test results

It can be seen that the purity of the obtained DNA is significantly lower than that of the extract obtained by the method of the present invention by using a conventional kit to extract a small amount of eye samples. Especially the OD280 value of the extract obtained by the control method is obviously higher than the OD280 value of the extract obtained by the method of the present invention. It shows that the DNA extraction method provided by the present invention can effectively separate DNA from other cell components and proteins. For the particularity of trace samples, the improvement of this step is the guarantee of high sensitivity and specificity of the detection method.

Example 2. Method for extracting DNA from eye trace solid specimens to be tested

Material:

Specimens to be tested: collected from admitted patients, including retina, corneal endothelium, pterygium, conjunctiva, iris, and eye tumors, with a collection volume of 1×1mm;

step:

(1) Put the collected sample to be tested into a container, add 10-30 μl proteinase K, 100-300 μl lysis buffer AL, and treat at 56°C for 6-12 hours;

(2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

(3) Put the liquid obtained in step (2) into a spin column, put it into a 2ml collection tube, and centrifuge at 6000-9000rpm for 0.5-2min;

(4) Add 300-600μl elution buffer 1, 6000-9000rpm, 0.5-2min, discard the filtrate and collection tube, and replace with a new collection tube;

(5) Add 300-600μl Elution Buffer 2, centrifuge at 10000-15000rpm for 1-5min, discard the filtrate and collection tube;

(6) Replace with a new 1.5ml liquid centrifuge tube, centrifuge at 10000-15000rpm, centrifuge for 0.5-2min, discard the filtrate and centrifuge tube;

(7) Change to a new 1.5ml centrifuge tube, add 50-100μl of elution buffer 3, place at room temperature for 0.5-2min, and centrifuge at 6000-9000rpm for 0.5-2min to obtain 50-100μl of DNA from the trace biological sample to be tested.

(8) Take 1 ul sample, and detect OD260 and OD280 by DNA content detection instrument nanodrop2000.

Control: Use a nucleic acid extraction kit (purchased from Daan Gene. As a control reagent, operate DNA extraction according to its instructions.

Test results:

It can be seen that the purity of the obtained DNA is lower than that of the extract obtained by the method of the present invention by using a conventional kit to extract a small amount of eye samples. The OD280 value of the extract obtained from conjunctival sac secretions and eye tumors is significantly higher than that of the present invention, that is, the protein or amino acid content in the extract is higher, and it is used as an amplification template, which easily affects the sensitivity of amplification. However, the method of the present invention can obtain high-purity templates for micro-samples of various tissues taken from the eye.

Example 3. Kit for detecting ocular VZV infection by ocular microsamples

DNA extraction reagent set:

Lysis buffer: Tris-saturated phenol with 10% SDS,

Elution buffer 1: a mixture of saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1;

Elution buffer 2: absolute ethanol,

Elution buffer 3: pH 8.0, 10 mmol/L Tris-HCl solution containing 1 mmol/LEDTA.

Specific primers and probes for PCR amplification

Primer-F: AACACCCGACTCGAAATACCA

Primer-R: TATTGGCACGCAACTCAACTG

Probe: AACTCAACTGGCACGCTTCG

Example 4. Kit for detecting ocular VZV infection by ocular microsamples

DNA extraction reagent set:

Lysis buffer: Tris-saturated phenol with 10% SDS,

Elution buffer 1: a mixture of saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1;

Elution buffer 2: absolute ethanol,

Elution buffer 3: pH 8.0, 10 mmol/L Tris-HCl solution containing 1 mmol/LEDTA.

Specific primers and probes for PCR amplification:

Primer-F: AACACCCGACTCGAAATACCA

Primer-R: TATTGGCACGCAACTCAACTG

Probe: AACTCAACTGGCACGCTTCG

PCR amplification control template:

Positive control template: the known VZV virus DNA fragment (contained with the control kit) is used to control the quality of the detection system and instruments, to ensure the accuracy of the results, and to avoid false negative results;

Positive control template: positive ocular fluid sample;

Weak positive control template: the diluent of positive ocular body fluid, and its critical value is set at ΔT=38-40.

Blank control template: ultrapure water;

Negative control template: Negative eye fluid sample;

Embodiment 5. Establishment of the detection method of VZV virus in the specimen

The samples were sera from patients who were clinically diagnosed with VZV infection in their blood, a total of 5 cases.

Step 1. Prepare sample DNA template by the method in Example 1

Step 2. Primer probe design and synthesis

Primer-F: AACACCCGACTCGAAATACCA

Primer-R: TATTGGCACGCAACTCAACTG

Probe: AACTCAACTGGCACGCTTCG

Step 3: Reaction system preparation

And set 5 control reaction tubes, the reaction system is the same as above, and the templates are:

Positive control template: known VZV virus DNA fragment control kit comes with

Positive control template: positive ocular fluid sample;

Weak positive control template: the diluent of positive ocular body fluid, and its critical value is set at ΔT=38-40.

Blank control template: ultrapure water;

Negative control template: Negative eye fluid sample;

Step 4. PCR program:

37℃2min

94℃2min

Cycle the above two temperatures and time, cycle 30-40 times

93°C for 15sec, 60°C for fluorescence detection

Comparison method:

Reagent:

Control extraction method: Use a nucleic acid extraction kit (purchased from Daan Gene. As a control reagent, operate DNA extraction according to its instructions.

Control detection method: The serum VZV detection kit commonly used in hospitals was used as the control method, purchased from Daan Gene, and the steps were the experimental steps included in the kit.

The test results are as follows:

In the above table, as the volume of the serum sample decreases to 10 microliters, the minimum detection limit of the method of the present invention for the liquid sample is 10ul; it can be seen that the detection accuracy of the detection method of the present invention is 4/5=0.80, which is 80%.

The control kit can only detect a 2ml serum sample volume, and as the sample volume decreases, meaningful test results cannot be obtained.

Example 6 Clinical Eye Specimen Detection Verification

The DNA comes from the eye specimens of clinically admitted patients, and the extraction method is shown in Example 1.

The PCR amplification method is the same as in Example 3.

Control method: Serum VZV detection kit commonly used in hospitals is used as a control method, purchased from Daan Gene, and the steps are the experimental operation steps attached to the kit.

The test results are as follows:

Figure 3 shows the detection result of one of the positive samples: the positive sample is similar to the weak positive control.

Example 7. There is a big difference between eye virus infection and blood virus infection

Samples: aqueous humor and blood from a clinically admitted patient at different stages

Detection method: the method of the present invention: the DNA comes from eye specimens and blood of clinically admitted patients, and the extraction method is shown in Example 1.

Control method: The serum VZV detection kit commonly used in the hospital was used as the control method, purchased from Daan Gene , and the steps were the experimental steps included in the kit.

Sample Test Results

Before the first stage of treatment: VZV was detected positive in eyes and venous blood;

The second stage: According to the conventional treatment method, the patient was given systemic anti-VZV virus drug - 1% prednisolone acetate for one month, and then changed to 0.1% Afluoride for two months. It showed that VZV in the blood turned negative after treatment, but systemic antiviral therapy was ineffective for ocular VZV infection.

The third stage: Three months later, the ocular specimen and venous blood were tested, and the ocular VZV infection turned negative.

The above data show that eye infection and blood infection are not synchronized. If only the blood infection is detected according to the current detection method commonly used in hospitals, and antiviral drugs are used for the whole body according to the detection results, it will not be effective for eye infection. Therefore, accurate detection of ocular infection is the key to effective treatment.

However, conventional methods and kits are used to detect ocular specimens, and no ocular VZV infection can be detected, so corresponding treatment cannot be performed on the eyes.

Treatment after detection

Sampling patients who are positive for VZV infection by the detection method of the present invention are given an individualized treatment plan for VZV, given intraocular local injections of antiviral drugs such as ganciclovir and acyclovir, and collected regularly after administration. Eye samples are tested for virus, and the virus DNA is detected after medication. According to the test results, the medication cycle and concentration are adjusted until the test results turn negative, as shown in Figure 1-2:

Figure 1. Intraocular viral endotheliitis

Figure 2. The eyeball shown in Figure 2 was confirmed to be VZV virus infection after virus detection. After a week of oral treatment with ganciclovir eye gel and acyclovir, the eyesight improved significantly and the vision recovered.

SEQUENCE LISTING

<110> Peking University Third Hospital

<120> Kit and method for detecting VZV infection in ocular trace biological samples

<130> P170785-BYS

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 21

<212>DNA

<213> Artificial sequence

<220>

<223> Primer-F

<400> 1

aacacccgac tcgaaatacc a 21

<210> 2

<211> 21

<212>DNA

<213> Artificial sequence

<220>

<223> Primer-R

<400> 2

tattggcacg caactcaact g 21

<210> 3

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> probe

<400> 3

aactcaactg gcacgcttcg 20

Claims (10)

1. a kind of for detecting the PCR amplification reagent of eye trace sample VZV infection, it is characterised in that: visited comprising following primer Needle group, sequence are as follows:

Primer-F:AACACCCGACTCGAAATACCA

Primer-R:TATTGGCACGCAACTCAACTG

Probe: AACTCAACTGGCACGCTTCG

The eye trace sample refers to that liquid sample of the volume no more than 10ul-200ul or volume are no more than the solid of 1 × 1mm Class sample；

The liquid sample refers to eye secretion, aqueous humor, tear and/or vitreum；

The solid tissue refers to the swollen object of retina, corneal endothelium, pteryium, conjunctiva, iris and/or eye.

2. PCR amplification reagent according to claim 1, which is characterized in that the PCR amplification system for each reaction contains Have:

The PCR amplification reagent makes reaction total volume reach 20 using preceding addition eye trace sample DNA profiling and distilled water A volume unit.

3. PCR amplification reagent according to claim 2, which is characterized in that the PCR amplification system for each reaction contains Have:

4. PCR amplification reagent according to claim 2 or 3, the volume unit be 1 microlitre, 1.5 microlitres, 2 microlitres or 2.5 microlitre.

5. a kind of for detecting the kit of eye VZV infection by eye trace sample, it is characterised in that:

Comprising any amplifing reagent infected for detecting eye trace sample VZV of claim 1-4, and

It is used to prepare the reagent set of eye trace sample DNA profiling, the reagent set includes following reagent；

Lysis buffer: the Tris saturated phenol containing 10%SDS,

Elution buffer 1: saturated phenol: chloroform: isoamyl alcohol is with the mixed liquor of volume ratio 25:24:1；

Elution buffer 2: dehydrated alcohol,

Elution buffer 3:pH8.0, the 10mmol/L Tris-HCl solution containing 1mmol/LEDTA；

The eye trace sample refers to that liquid sample of the volume no more than 10ul-200ul or volume are no more than 1mm³Solid kind sample This；The liquid sample refers to eye secretion, aqueous humor, tear and/or vitreum；The solid tissue refers to retina, in cornea The swollen object of skin, pteryium, conjunctiva, iris and/or eye.

6. kit according to claim 5, it is characterised in that: be also included as each detection reaction setting control reaction Template is compareed, specific as follows:

Positive control template: known VZV viral dna fragment guarantees result for the quality control to detection architecture and instrument Accuracy avoids false negative result

Positive control template: positive eye body fluid；

Blank control template: ultrapure water；

Negative control template: negative ocular fluids sample；

Weakly positive compares template: the dilution of positive eye body fluid, and critical value is arranged in △ T=38-40.

7. a kind of for detecting the PCR method of eye VZV infection by eye trace sample, which is characterized in that

Including preparing eye trace sample DNA profiling, then using any PCR amplification reagent of claim 1-4 to institute It states eye trace sample DNA profiling and carries out real-time quantitative PCR detection；

Described the step of preparing eye trace sample DNA profiling, is as follows:

(1) micro-biological sample to be measured is put into container, 10-30 μ l Proteinase K, 100-300 μ l lysis buffer AL is added, It is handled at least 10 minutes in 56 DEG C；The micro-biological sample refers to liquid sample of the volume no more than 10ul-200ul or volume not Solid kind sample more than 1 × 1mm；

(2) it is added and mixes 10-20s, brief centrifugation with the isometric dehydrated alcohol of lysis buffer, concussion；

(3) step (2) resulting liquid is put in centrifugal column, is put into 2ml collecting pipe, 6000-9000rpm is centrifuged 0.5- 2min；

If 140 μ l of sample size >, repeat the above steps (3)；

(4) 300-600 μ l elution buffer 1,6000-9000rpm, 0.5-2min is added, abandons filtrate and collecting pipe, the receipts renewed Collector；

(5) 300-600 μ l elution buffer 2,10000-15000rpm is added, is centrifuged 1-5min, abandons filtrate and collecting pipe；

(6) the 1.5ml liquid centrifuge tube renewed, it is empty to be centrifuged 0.5-2min from 10000-15000rpm, abandon filtrate and centrifuge tube；

(7) a new 1.5ml centrifuge tube is changed, 50-100 μ l elution buffer 3 is added, is placed at room temperature for 0.5-2min, is centrifuged 6000- 9000rpm, 0.5-2min obtain the DNA profiling of micro-biological sample to be measured；

Wherein:

The lysis buffer is the Tris saturated phenol containing 10%SDS；

The formula of the elution buffer 1 are as follows: saturated phenol: chloroform: isoamyl alcohol is with the mixed liquor of volume ratio 25:24:1；

The elution buffer 2 is dehydrated alcohol；

The elution buffer 3 is pH8.0, the 10mmol/L Tris-HCl solution of the EDTA containing 1mmol/L；

The eye trace sample refers to that liquid sample of the volume no more than 10ul-200ul or volume are no more than 1mm³Solid kind sample This；The liquid sample refers to eye secretion, aqueous humor, tear and/or vitreum；The solid tissue refers to retina, in cornea The swollen object of skin, pteryium, conjunctiva, iris and/or eye.

8. PCR method according to claim 7, which is characterized in that described the step of preparing eye trace sample DNA profiling It is as follows:

(1) micro-biological sample to be measured is put into container, 20 μ l Proteinase Ks, 200 μ l lysis buffers is added, at 56 DEG C Reason at least 10 minutes；

(2) it is added and mixes 10-20s, brief centrifugation with the isometric dehydrated alcohol of lysis buffer, concussion；

(3) step (2) resulting liquid is put in centrifugal column, is put into 2ml collecting pipe, 6000-9000rpm is centrifuged 0.5- 2min；If 140 μ l of sample size >, repeat the above steps (3)；

(4) 500 μ l elution buffers 1,8000rpm are added, are centrifuged 1min, abandon filtrate and collecting pipe, the collecting pipe renewed；

(5) 500 μ l elution buffers 2,14000rpm are added, are centrifuged 3min, abandon filtrate and collecting pipe；

(6) the 1.5ml liquid centrifuge tube renewed, it is empty to be centrifuged 1min from 14000rpm, abandon filtrate and centrifuge tube；

(7) 50-100 μ l elution buffer 3 is added in the 1.5ml centrifuge tube renewed, is placed at room temperature for 1min, is centrifuged 8000rpm, 1min obtains the DNA of micro-biological sample to be measured.

9. PCR method according to claim 7 or 8, which is characterized in that when the micro-biological sample to be measured is liquid, It in 56 DEG C of processing 10min and then is put into 70 DEG C of environment in step (1) and handles 10min；

When the survey micro-biological sample is solid tissue, in 56 DEG C of 5-12 hours of processing in step (1).

10. according to any PCR method of claim 7-9,

The program of PCR reaction is as follows:

37℃×2min

94 DEG C × 2min, above-mentioned two temperature and time recycles 40 times

93 DEG C × 15sec,

60 DEG C of detection fluorescence.