Wuxi Shenrui Bio-Pharmaceuticals Co., Ltd. [China]

Human Papillomavirus (high risk subtype) Nucleide Detection System Kit (PCR-fluorescent probe method)

Human Papillomavirus (high risk subtype) Nucleide Detection System Kit (PCR-fluorescent probe method) Instruction Manual

Name: Human papillomavirus (high risk subtype) nucleide detection system kit (PCR-fluorescent probe method)

Specification: 24 pieces/ box or 48 pieces/ box

Type: the kits are divided into Type I and Type II:

a)       Type I can detect whether it’s single or compound HPV high risk subtype infection, but it can’t confirm which type or types it is or they are.

b)       Type II can detect whether it’s single or compound HPV high risk subtype infection, and it can judge whether it’s Type 16 or Type 18.

Expected purpose: It’s used to qualitatively detect 14 high risk subtypes (HPV16,18,31,33,35,39,45,51,52,56,58,59,66,68) of human papillomavirus DNA in women’s cervical cells.

Principle of test: Based on human papillomavirus DNA as the template, it takes use of PCR fluorescent method to detect the infection condition.

Major composition:

Storage condition and validity: it should be preserved under temperature of -18’C~ -20’C, kept in darkness. The validity is 12 months , please use the kits in validity.

Applicable instruments: ABI7500, Bio-Rad CFX96, Roche LC480 etc.

Specimen requirements:

1, Preparation before test:

1)       Menstruation is normal and the test should be taken 10-18 days after menses for the best results.

2)       No sexual behavior in 24 hours before test.

3)       No vinegar or iodine solution smeared before test.

4)       No drug used or washed in the vagina in 72 hours before test , or it’ll influence the test results.

2, Specimen collection method and steps:

1)       Gently wipe up the excessive cervical secretion with cotton swabs. The cytobrush should be close to the cervical mucosa. And it should be clockwise rotated for 3-6 turns to collect the enough deciduous cervical cells.

P.s. Please strictly follow the above specimen collection steps, the quantity of the deciduous cervical cells will directly affect the accuracy of testing results.

2) Carefully take out the cytobrush and put it into specimen saving tube which is equipped with 3ml sterile Isotonic phosphate buffer. The deciduous cervical cells attached with the cytobrush should be wholly washed into the tube. Then the hair of cytobrush should be well wiped and thrown away. The tube cap should be screwed down, the tube should be labeled with patient’s number, and then be delivered to inspection.

Testing method:

1, DNA extraction:

Take 1ml sterile Isotonic phosphate buffer with deciduous cervical cells out (if the quantity of cells is small, the volume of the liquid should become 2ml), and transfer the liquid into a new centrifugal tube.

Put the tube into a centrifugal machine, rotated with a speed of 10,000~ 13,000    turns/second, to be centrifuged for 10 mins, and then abandon the liquidsupernatant. The deciduous cervical cells will form sediment on the bottom of the tube(the amount of the sediment doesn’t indicate the quantity of deciduous cervical cells).

Add 50ul Virus cracking liquid into the tube, shake and mix it up , heat the tube up till boiled for 10 mins. Resuspend the deciduous cervical cells. The cells should be sufficiently resuspended by a Whirlpool blending oscillator so that the quantity of DNA will be more.

Put the compound liquid of step 3 in a centrifugal machine, rotated in a speed of 10,000 ~ 13,000 turns/ min, to be centrifuged for 10 mins. The liquidsupernatant is regarded as the template of PCR reaction now.

Take 2ul PCR reaction template and the rest is preserved under temperature of -20’c.

2, Add specimen into DNA template

Carefully add 2ul PCR reaction template into PCR reaction tube. We suggest you should set up negative and positive contol groups for each test. You should add 2ul positive and negative quality control liquid into relative PCR tubes.

3, Preparation of amplification reagents:

Unfreeze the reagents. Take out the Human papillomavirus (high risk subtype) nucleide detection system kit and unfreeze it until it returns to the room temperature.

Centrifuge the reagents. Make sure the reagents attached with the cap and tube have been centrifuged to the bottom of the tube and mixed up.

Allocate the PCR reaction premix compound liquid as the following table required. And then put the liquid in the centrifugal machine and rotate it, make sure the eagents attached with the cap and tube have been centrifuged to the bottom of the tube and mixed up.(due to the loss caused by adding the specimen, we suggest that you should prepare one more piece of premix liquid for every 10 pieces.)

Allocate the PCR reaction premix liquid into the reaction holes of the existing templates as 23ul per capita. And seal the fluorescent qualitative PCR level sealing film or screw up the cap and rotate with a speed of 2,000 turns/min, centrifuge for 30s. And make sure the liquid has been centrifuged into the bottom.

4, PCR reaction procedure

Set up the reaction conditions as following table required based on your PCR instrument.

Instruction of fluorescent testing channels:

Analysis of testing results:

Analyze the results based on the different softwares of instruments, and get the HPV DNA testing results.(the baseline is selected as 6~10loops or 6~15loops, threshold line is selected as the peak of negative comparison normal amplification curve). The clinical specimen judgment should follow the following steps:

1, quality controlling standards

The positive comparison testing result is positive, and Ct Value is no more than 35.

The negative comparison testing result is negative(Ct Value has countless values).

Inner reference gene’s Ct value is no more than 38 (if the inner reference of specimen’s Ct value is more than 38, the specimen is positive, the report should be positive; if the specimen is negative, we suggest that you should retest it.)

The above 3 conditions should be totally satisfied, or the test is invalid.

2, result judgment

If the Ct value has countless values, the specimen is negative.

The specimen whose Ct value is no more than 40 is positive.

When the Ct value is between 40 and 45, we suggest you should retest it. If the result of retest is less than 40, it’s positive, or it’s negative.

The limitation of the testing method:

1, the testing result of the testing kit is only provided as a clinical reference. The treatment of the patients should be incorporated with other clinical data.

2, possibility analysis about false negative result

It’ll be caused by unreasonable specimen collection, transferring and treatment, as well as the virus quantity in specimen is too low.

It’ll be caused by the mutation of the target sequence of human papillomavirus attempting to test or mutations caused by other reasons.

It’ll be caused by other unverified interferences or PCR inhibiting factors.

Product performance indicators:

Sensitivity: the testing kit’s sensitivity should be no less than 500 copies per reaction.

Stability: the kits should be preserved under temperature of -18C~ -20C. The validity is 12 months.

Bottle stability: if the kit hasn’t run out, please preserve the kit under temperature of -18C~ -20C, with cap screwed up and kept in darkness. The kit is valid in validity.

Precautions:

1, if the specimens are not for testing right now, please preserve them under temperature of -18C~20C.

2, the components of the kit should be unfrozen and mixed up before using, and be well centrifuged.

3, the experimental area should be divided into different parts, the stuff in different parts should not be cross used. Each part should have their exclusive instruments and equipment.

4, the regulation of the lab should be in referenced to the genetic amplification lab regulation issued by relative bureaus.

5, PCR operators should be professionally trained.

6,the instruments used in operation processes should be sterilized regularly by 10% hypochlorous acid or 75% ethanol. The experimental area should be ultraviolet sterilized regularly. The abandoned suction nozzles should be thrown into disposal bottles(10% hypochlorous acid inside) to prevent the contamination.

7, please operate by the regulation of communicable diseases lab.

References:

1, Instruction of Medical Appliances, Regulation of Labels and Packaging Signatures(issued by SFDA, China, July 8th, 2004)

2, Notice About Issuing The Guidelines Of IVD reagents clinical studying technology and The Guidelines Of IVD reagents instruction composition.(SFDA, China, April 1st, 2011)

The Manufactory: Wuxi Shenrui Bio-Pharmaceuticals Co., Ltd

Add: No.88, West Meiliang Rd., (Bio-Park), Binhu District, Wuxi City, China

Phone Number: +86 510 8599 8839

Fax: +86 510 8599 9325

Hotline: 400 686 3389

Medical Equipment Production License: S2004-0017

Website: www.sr-bio.com

Human Papillomavirus (high risk subtype) Nucleide Detection System Kit (PCR-fluorescent probe method)