Measles Virus and Rubella Virus Nucleic Acid Detection Kit (Fluorescent PCR Method)

Instruction Manual for the Measles Virus and Rubella Virus Nucleic Acid Detection Kit (Fluorescent PCR Method)
[Product Name]
Generic Name: Measles Virus and Rubella Virus Nucleic Acid Detection Kit (Fluorescent PCR Method)
[Packaging Specifications] 10 servings/box, 20 servings/box, 50 servings/box.
[Intended Use]
This product is intended for the qualitative detection of measles virus (MV) and rubella virus (RV) nucleic acids in pharyngeal swab samples.
Measles virus is a single-stranded negative-sense RNA virus belonging to the genus Morbillivirus within the family Paramyxoviridae. Measles is an acute respiratory infectious disease caused by the measles virus, clinically characterized by fever, rash, and cough.
Rubella virus is the sole member of the genus Rubivirus within the family Togaviridae; it is a single-stranded positive-sense RNA virus. Rubella is an acute febrile exanthematous infectious disease caused by rubella virus, clinically characterized by fever and rash, and can result from either congenital or acquired infection. Clinically, it is marked by a short prodromal period, low-grade fever, a maculopapular rash, and enlargement of postauricular and occipital lymph nodes.
Measles and rubella are both respiratory‑borne infectious diseases characterized by acute fever and a rash; they frequently cause epidemics or outbreaks, posing a serious threat to the health of children and adolescents. Clinically, rubella, measles, and other febrile exanthematous illnesses are difficult to distinguish. At present, the main laboratory diagnostic methods include virus isolation, serological assays, and nucleic acid testing.
[Principle of Assay]
This product employs real-time fluorescent PCR technology, using conserved regions of the measles virus and rubella virus genomes as amplification targets. Specific primers and probes are designed for PCR amplification, enabling qualitative detection of whether a sample contains nucleic acids of either measles virus or rubella virus within a single reaction system. The product incorporates an internal control assay to monitor nucleic acid extraction, reverse transcription, and PCR amplification, thereby minimizing the risk of false-negative results.
[Main Ingredients]
Component Name Main Ingredient Specification and Quantity (Unit: vials)
10 servings/box, 20 servings/box, 50 servings/box
Reaction Solution A: primers, probe, dNTPs, Mg²⁺, K⁺. 180 μL × 1, 360 μL × 1, 900 μL × 1
Reaction Solution B: reverse transcriptase, DNA polymerase, RNase inhibitor, glycerol. 35 μl × 1, 65 μl × 1, 160 μl × 1
Positive control: plasmids containing measles virus– and rubella virus–specific fragments. 20 μl × 1, 50 μl × 1, 100 μl × 1
Negative control: DEPC-treated purified water. 500 μL × 1, 500 μL × 1, 500 μL × 1
[Storage Conditions and Shelf Life]
Store protected from light at 20±5℃; shelf life: 12 months.
The number of freeze–thaw cycles should not exceed 10, and once opened, the product should be used within three months when stored at −20°C. The manufacturing date and expiration date are indicated on the label.
[Applicable Instruments]
ABI 7500, Stratagene Mx3000P, and Bio-Rad CFX96 real-time PCR instruments.
[Sample Requirements]
The applicable specimen type is a throat swab.
Sample Collection: Slightly moisten a sterile cotton swab with physiological saline, then repeatedly swab the patient’s pharynx several times. Subsequently, immerse the swab in 1–2 mL of specimen‑preserving solution (containing 500–1,000 units/mL penicillin, 500–1,000 μg/mL streptomycin, and 2% bovine serum in DMEM) or in viral transport medium (VTM), gently squeeze the swab several times, and discard the swab.
Samples should be tested promptly after collection; they can be stored at −20°C ± 5°C for up to 6 months, and at −70°C for long-term preservation. Avoid repeated freeze–thaw cycles; if unavoidable, limit the number of cycles to no more than five, and use low-temperature shipping.
[Testing Method]
1. Sample Processing (Sample Preparation Area)
RNA was extracted using the QIAamp Viral RNA Mini Kit from QIAGEN (Catalog No.: 52904) or our company’s nucleic acid extraction kit (Catalog No.: HT5025; Registration No.: Yue Zhu Xie 20140038), following the manufacturer’s instructions.
2. PCR Reagent Preparation (Reagent Preparation Area)
Remove Reaction Solution A and Reaction Solution B from the kit; allow them to thaw at room temperature, then vortex thoroughly to mix. Centrifuge briefly for a few seconds before use.
Take N PCR reaction tubes (where N = the number of samples to be tested + negative control + positive control), and prepare the reaction mixture for each tube as shown in the table below:
Reaction Solution Components Amount Added (μl) per Sample
Reaction Solution A 17
Reaction Solution B 3
Total volume 20
After thoroughly mixing all components, centrifuge briefly for a few seconds.
3. Sample Addition (Sample Preparation Area)
Add 5 μL of the extracted RNA sample to be tested, along with 5 μL of the negative control and 5 μL of the positive control, into the corresponding reaction tubes. Securely cap the tubes, briefly centrifuge, and then transfer them to the amplification and detection area.
4. PCR Amplification Assay (Amplification Detection Zone)
4.1 Place the reaction tube into the fluorescence PCR instrument for amplification and detection.
4.2 Cycle Parameter Settings (Please refer to the operating instructions for each instrument when configuring.)
Step Temperature Time Number of Cycles (times)
1 Reverse transcription reaction 47°C 30 min 1
2 Pre-denaturation 95℃ 10 min 1
3 Denaturation 95℃ 10s 45
4 Annealing, extension, and fluorescence collection 56°C 45 s
In Step 4, fluorescence detection is performed at 56°C. The ROX/Texas Red channel serves as the internal reference, the FAM channel detects measles virus, and the HEX/VIC channel detects rubella virus.
On the ABI 7500 real-time PCR instrument, do not select ROX correction, and set the quencher to None.
4.3 Results Analysis
After the reaction is complete, adjust the Baseline’s Start and End values as well as the Threshold value, then click “Analysis” to automatically obtain the analysis results.
[Positive Judgment Value or Reference Interval]
The receiver operating characteristic (ROC) curve was used to determine the positive cutoff values for this kit: Ct < 38 for measles virus and Ct < 38 for rubella virus.
[Interpretation of Test Results]
Quality Control
1. Negative control: No typical S-shaped amplification curve is observed;
2. Positive control: Both the FAM and HEX detection channels exhibit typical S-shaped curves with Ct values ≤ 30, while the ROX detection channel shows…
Or no amplification curve;
3. The above requirements must be met simultaneously in a single experiment; otherwise, the experiment will be deemed invalid and must be repeated.
Result Interpretation
1. Interpretation of negative results:
If the FAM and HEX detection channels show no amplification curve or a Ct value ≥40, while the ROX detection channel exhibits an amplification curve, the sample can be classified as negative for measles virus and rubella virus.
2. Interpretation of Positive Results:
If the FAM detection channel shows an amplification curve with a Ct value less than 38, and the ROX detection channel either has or does not show an amplification curve, the sample is considered positive for measles virus.
If the HEX detection channel shows an amplification curve and the Ct value is less than 38, and the ROX detection channel either has or does not have an amplification curve, the sample can be classified as positive for rubella virus.
3. Interpretation of Suspicious Results:
If the FAM detection channel shows an amplification curve and 38 ≤ Ct < 40, while the ROX detection channel may or may not show an amplification curve, the sample must be retested. If, upon retesting, the FAM Ct value remains within the 38–40 range and exhibits a clear exponential growth phase, the sample is considered positive for measles virus; otherwise, the sample is deemed negative for measles virus.
If the HEX detection channel shows an amplification curve and 38 ≤ Ct < 40, while the ROX detection channel either has or lacks an amplification curve, the sample must be retested. If, upon retesting, the HEX Ct value remains within the 38–40 range and exhibits a clear exponential growth phase, the sample is considered positive for rubella virus; otherwise, it is deemed negative.
[Limitations of the Testing Method]
1. Errors in sample collection, processing, transportation, and storage can all lead to inaccurate results.
2. Cross-contamination during sample processing may lead to false-positive results.
3. Genetic mutations in the virus may also lead to false-negative results;
4. Due to the large number of primers and probes used in this method, primer dimers may form, leading to false-positive results.
[Product Performance Specifications]
1. Limit of detection: 5 TCID50/mL for both measles virus and rubella virus.
2. Precision: Intra-assay coefficient of variation ≤ 5%, inter-assay coefficient of variation ≤ 5%.
3. Specificity: This product shows no cross-reactivity with dengue virus, Japanese encephalitis virus, varicella-zoster virus, mumps virus, influenza virus, hepatitis B virus, hepatitis C virus, HIV, Toxoplasma gondii, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, or respiratory syncytial virus. No experimental data are currently available regarding reactivity with other pathogens.
4. Subtype detection capability: The assay can detect measles virus genotypes H1a, A, B3, D8, and D9; rubella virus genotypes 2B and 1E; no experimental data are currently available for other genotypes.
5. Interference Testing: Common interfering substances—such as penicillin, dexamethasone, vitamin C, acetaminophen, blood, pus, and vidarabine—at concentrations of 20 μg/mL, 0.3 μg/mL, 30 μg/mL, 20 μg/mL, 25%, 25%, and 200 mg/mL, respectively, do not affect the assay results.
6. In clinical trials, this product was tested on 596 throat swab samples and compared with the reference method:
For measles detection, the sensitivity was 100%, the specificity was 95.7%, and the overall concordance was 97.5%.
For rubella testing, the sensitivity was 100%, the specificity was 97.1%, and the overall agreement was 97.6%.
[Precautions]
1. This product is an in vitro diagnostic reagent; operators must undergo professional training and possess relevant experience.
2. To ensure the accuracy and reliability of experimental results, please use calibrated pipettes. All laboratory consumables (e.g., disposable PCR tubes, tips, centrifuge tubes) must be RNase‑free.
3. The experiment must be conducted in strictly segregated zones (reagent preparation, sample preparation, and amplification–detection). Each zone must use dedicated equipment and work attire; cross‑contamination is prohibited, and all surfaces must be promptly cleaned after the experiment.
4. Sample handling must be performed within a biosafety cabinet to ensure operator safety and prevent environmental contamination.
5. RNA samples should be stored at −70°C; prior to aliquoting, they must be fully thawed at room temperature, thoroughly mixed, and briefly centrifuged before use.
6. Each experiment must include both a negative control and a positive control. Do not mix reagents from different lot numbers, and use the kit within its expiration date.
7. After mixing MV/RV PCR Reaction Solution A with MV/RV PCR Reaction Solution B, the experiment should be completed as soon as possible; otherwise, the amplification efficiency will decline over time.
8. During the experiment, wear a disposable mask and a disposable hair cap, and frequently change your disposable plastic gloves.
9. When handling PCR tubes, never wear latex gloves to prevent sweat or powder from the gloves from interfering with fluorescence measurements.
10. After adding the template to the reaction mixture, invert and mix thoroughly, gently tap the tube wall to remove air bubbles, and briefly centrifuge.
11. Used pipette tips shall be immersed in a waste container containing 10% sodium hypochlorite and, after the experiment, disposed of together with other waste materials.
12. After amplification is complete, carefully remove the PCR tubes and place them in a sealed bag. Opening the tubes in the PCR laboratory is strictly prohibited; if opened, aerosols could contaminate the PCR room, leading to false-positive results.
13. After the experiment, treat the workbench and pipettes with 10% sodium hypochlorite or 75% ethanol, then expose them to UV light for 30 minutes.
14. The test samples involved in this product should be treated as infectious materials, and direct contact with the body should be avoided. All sample handling and processing must comply with the Ministry of Health’s “General Biosafety Guidelines for Microbiological and Biomedical Laboratories” and the relevant requirements of the “Regulations on the Administration of Medical Devices.”
15. The internal control detection system of this product is used to evaluate the efficiency of sampling and extraction only when both rubella virus and measles virus tests are negative. If either rubella virus or measles virus is positive, the presence or absence of the internal control does not affect the interpretation of the results.
[Explanation of the Logo]
                                                
Temperature limits In vitro diagnostic medical devices Refer to the instructions for use Do not reuse
[References]
Mosquera Mdel M, de Ory F, Moreno M, Echevarria JE. Simultaneous detection of measles virus, rubella virus, and parvovirus B19 by using multiplex PCR. J Clin Microbiol, 2002, 40(1): 111-116.
[Basic Information]
Registrant/Manufacturer Name: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Address: No. 18, Innovation Road No. 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City Postal Code: 519085
Contact Information: Telephone: 0756-3890858 Fax: 0756-3890848
Production Address: No. 18, Innovation Road 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City Postal Code: 519085
Production License No.: Yue Food and Drug Administration Medical Device Production License No. 20040941
[Medical Device Registration Certificate Number/Product Technical Requirements Number] National Medical Device Registration No. 20183400480
[Date of Approval and Date of Revision of the Instruction Manual] 2018.11.07