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Kit and method for detecting CMV infection in trace biological sample of eye

A biological sample and ocular technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of inability to accurately reflect the real cause of the eye and the lack of high-sensitivity detection of viral infection in ocular tissue Means, unseen issues, etc.

Active Publication Date: 2018-12-07
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another example is the high incidence of CMV infection in patients with tumor transplantation. At present, the clinical focus is only on blood testing and systemic administration. Due to the lack of intraocular detection methods, intraocular CMV infection is often overlooked, and secondary CMV intraocular infection occurs. Cases leading to vision loss are also increasing
[0004] At present, blood tests are still used to detect ocular viral infections, but 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 easy to miss and misdiagnose
[0005] 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. Reports of High Sensitivity Detection of Viral Infections by PCR

Method used

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  • Kit and method for detecting CMV infection in trace biological sample of eye
  • Kit and method for detecting CMV infection in trace biological sample of eye
  • Kit and method for detecting CMV infection in trace biological sample of eye

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1. Method for extracting DNA from test specimens of eye microfluidics

[0118] Material:

[0119] Specimens to be tested: collected from admitted patients, tear fluid, aqueous humor 2, and vitreous.

[0120] (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;

[0121] (2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

[0122] (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;

[0123] If the sample volume > 140 μl, repeat step (3);

[0124] (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;

[0125] (5) Add 300-600μl Elution Buffer 2, centrifuge at 10000-15000rpm for 1-5min, discard the filtrate and collection tu...

Embodiment 2

[0134] Example 2. Method for extracting DNA from eye trace solid specimens to be tested

[0135] Material:

[0136] 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;

[0137] step:

[0138] (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;

[0139] (2) Add the same volume of absolute ethanol as the lysis buffer, shake and mix for 10-20s, and centrifuge briefly;

[0140] (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;

[0141] (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;

[0142] (5) Add 300-600μl Elution Buffer 2, centrifuge at 10000-15000rpm for 1-5min, discard th...

Embodiment 3

[0150] Example 3. Kit for detection of ocular CMV infection by ocular microsamples

[0151] DNA extraction reagent set:

[0152] Lysis buffer: Tris-saturated phenol with 10% SDS,

[0153] Elution buffer 1: a mixture of saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1;

[0154] Elution buffer 2: absolute ethanol,

[0155] Elution buffer 3: pH 8.0, 10 mmol / L Tris-HCl solution containing 1 mmol / LEDTA.

[0156] Specific primers and probes for PCR amplification

[0157] Primer-F: TCGTGAAACACGCTCGCTTT

[0158] Primer-R: TGGTCGGTACACGGTCCCTT

[0159] Probe: GACCCGGTGTTCCTTGGAGA

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Abstract

The invention relates to a kit and a method for detecting CMV infection in a trace biological sample of an eye, and relates to a molecular detection technology for eye viral infections, in particularto a method for extracting DNA from the trace biological sample, wherein the trace biological sample refers to a liquid sample with a volume not exceeding 10mu l-200mu l or a solid sample with a volume not exceeding 1mm<3>. By use of the extracted DNA as a PCR detection template, the eye viral infections can be detected with an accuracy of 80% or more, and the method provides a reliable basis forsubsequent appropriate treatment methods and reduces the blindness of the treatment methods.

Description

technical field [0001] The invention relates to a molecular detection technology of virus infection, in particular to a kit and a method for detecting CMV infection in eye trace biological samples. technical background [0002] 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 extrac...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6806C12Q1/6851C12N15/11
CPCC12Q1/6806C12Q1/6851C12Q1/705C12Q2527/125C12Q2531/113C12Q2545/113
Inventor 张培冯丽娜黄琛王薇
Owner PEKING UNIV THIRD HOSPITAL
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