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Kit for Rapid Enrichment of SARS Virus

Brief Introduction

During incubation period and the course of disease, SARS virus replicates and releases from cells. The level of virus varies greatly from sample to sample. At early stage of incubation period or during the late convalescent phase, the concentration of virus is very low in all kinds of samples. Virus is also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. It is very important for prevention and therapy to detect virus as early as possible. However, so far commercially available kits can only isolate viral nucleic acid from small volume of sample so that the positive rate for SARS assay is still low. Obviously,it is necessary to enrich virus from samples with low level of virus in order to increase the sensitivity of viral detection by RT-PCR or immunoassay. The Kit for Rapid Enrichment of SARS Virus is designed to enrich and partly purify SARS virus from clinical samples such as gargling liquid, cell culture supernatant, urine, plasma, stool extract, sputum extract and extract from nasal swab and throat swab. The Kit can also remove RT-PCR inhibitors, and increases the concentration of viral RNA before RT-PCR, which will greatly improve detection sensitivity and is useful for early diagnosis.

Kit Contents

Cat. No. 115010-25    

Price: 435 Euro/ kit

Preparation 25 samples

Buffer PC-A  60 ml

Buffer PC-B  100 ml

Buffer VP  135 ml

Manual 1

All buffers can be stored at room temperature (15~25?) for at least two years without showing reduction in the efficiency of purification.

Buffer PC-A: Virus co-precipitant. Store at room temperature.

Buffer PC-B: Phase partition buffer. It can also kill virus. Store at room temperature.

Buffer VP: Virus precipitation buffer. Store at room temperature.

 Principle

Clinical sample is added to Buffer PC-A, which contains high concentration of salt to dehydrate and salt out virus. After addition of Buffer PC-B, two phases are formed and virus is precipitated on the interface. Discard part of the lower-phase, and keep the precipitate containing virus on the interface. Add Buffer VP to form single phase and precipitate virus to the bottom of tube by centrifugation. Thus, the resulting virus pellet is enriched and partially purified, and can be used for viral RNA purification immediately. Buffer PC-A and Buffer PC-B are strong RNase inhibitors, which can protect viral RNA from degradation. Additionally, Buffer PC-B can kill virus so that the risk of the laboratory contamination is greatly reduced.

Notes

1. SARS virus is highly infectious and will cause severe acute respiratory syndrome in humans. Operators should be protected with essential defense measures before contact with clinical samples.

2. All operation steps should follow instruction strictly. The waste should be placed in the vessels containing disinfectant.

3. All apparatus used in the experiment should be free from contamination with nucleic acid, and attention should be paid to avoid laboratory contamination with PCR products.

Preparation Before Experiment

The following preparation is needed for all clinical samples: 1. Vessel containing disinfectant. 2. Pipette. 3. Chill Buffer PC-B at 4?. 4. Prepare 50% ethanol and chill at 4?. 5. Chill the chamber of centrifuge at 4?.

The following lab-wares and materials are required independently for different clinical specimens:

1. Gargling liquid, cell culture supernatant, urine: 50-ml Centrifugation Tube.

2. Plasma: 15-ml Centrifugation Tube.

3. Sputum: 50-ml Centrifugation Tube; 0.9% NaCl or PBS?, in which add N-Acetylcystein to final concentration of 1% just before use.

4. Nasal swab: 15-ml Centrifugation Tube, 0.9% NaCl or PBS?.

5. Throat swab: 15-ml Centrifugation Tube and 50-ml Centrifugation Tube, 0.9% NaCl or PBS?.

6. Stool: 50-ml Centrifugation Tube, 0.9% NaCl or PBS*.

*: PBS:1.44 g Na2HPO4·2H2O (or 1.15 g Na2HPO4), 0.2 g KH2PO4, 0.2 g KC1,8.0 g NaCl,add water to 1 litter, adjust pH value to 7.2 and autoclaved or filtrated through 0.22 m membrane.

Protocol

The protocol is designed to enrich virus from the following clinical specimens: A. 5 ml of gargling liquid, cell culture supernatant, urine and stool extract; B. 2 ml of plasma, sputum extract and extract from throat swab or nasal swab. To enrich virus from specimens with other volume, all extraction buffers should be added in proportion. The ratio of sample and extraction buffers goes as follows: sample volume/ Buffer PC-A/ Buffer PC-B/ Buffer VP = 5/ 2/ 6.5/5.

[Protocol for Enrichment of SARS virus from gargling liquid, cell culture supernatant, urine and stool extract]

bulletbullet 1. Collect clinical sample and release virus

A. Gargling liquid, cell culture supernatant and urine Apply 5 ml of sample in 50-ml Centrifugation Tube, add 2 ml of Buffer PC-A.

B. Fresh stool (1) Weigh 2 g of sample and apply in 50-ml Centrifugation Tube.

bulletbullet 2. Add 6 ml of 0.9% NaCl or PBS, and agitate to break it with pipette. Suspend it completely by pipetting solution in and out repeatedly to release the virus completely (Note: avoid spattering outside). Centrifuge at 5000×g for 5 min at 4?. (3) Transfer 5 ml of supernatant to 50-ml Centrifugation Tube. Add 2 ml of Buffer PC-A? 2. Add 6.5 ml of Buffer PC-B pre-chilled at 4?. Pipette to mix well, incubate at room temperature for 5 min and then shake vigorously.
 
* Buffer PC-B can kill virus. In order to avoid contaminating laboratory, do not shake vigorously but pipette to mix well after addition of Buffer PC-A and Buffer PC-B. Stand for 5 min to kill virus, and then shake vigorously to mix well.
 
bulletbullet 3. Centrifuge at =5000×g for 5 min at 4? to precipitate virus and cells on the interface. 4. Add 5 ml of Buffer VP and mix well, centrifuge at =5000 g for 3 min at 4? to pellet virus and cells on the bottom of tube. 5. Discard the supernatant. Add 5 ml of 50% ethanol, and rotate the tube to wash off the solution on the wall. Centrifuge at =5000 g for 3 min at 4?. 6. Discard the supernatant, short spin and remove all solution.
* The virus pellet should be used for purification of viral RNA immediately, or store at -20?.

Protocol for Enrichment of SARS virus from plasma, sputum extract, and extract from nasal swab or throat swab:

1. Collect clinical specimens and release virus

A. Plasma

Apply 2 ml of sample in 15-ml Centrifugation Tube, add 0.8 ml of Buffer PC-A.

B. Sputum

1. Collect sample and apply in 50-ml Centrifugation Tube.

2. Add 2.5 ml of 0.9 % NaCl or PBS containing 1% acetylcysteine, vortex for 3~10 min to release the virus completely (the time of vortex is dependent on the viscosity of sputum). Centrifuge at 10000×g for 5 min at 4?.

 3. Transfer 2 ml of supernatant to a new 15-ml Centrifugation Tube. Add 0.8 ml of Buffer PC-A?

C. Extract from nasal swab

(1) Apply nasal swab in 15-ml Centrifugation Tube.

(2) Add 3 ml of 0.9% NaCl or PBS, close the cap of centrifugation tube tightly. Vortex for 1 min to release virus completely. Clip the nasal swab, slightly squeeze out the residual extraction solution in the swab, and take it out carefully. Do not contaminate the outlet of the tube, and discard it into the vessel containing disinfectant.

(3) Transfer 2 ml of extract into a 15-ml centrifugation tube. Add 0.8 ml of Buffer PC-A.

D. Extract from throat swab

(1) Apply nasal swab in 50-ml Centrifugation Tube.

(2) Add 3 ml of 0.9% NaCl or PBS, close the cap of centrifugation tube tightly. Vortex for 1 min to release virus completely. Clip the nasal swab, slightly squeeze out the residual extraction solution in the swab, and take it out carefully. Do not contaminate the outlet of the tube, and discard it into the vessel containing disinfectant.

(3) Transfer 2 ml of extract into a 15-ml centrifugation tube. If the supernatant is less than 2 ml, make it up to 2 ml with 0.9% NaCl or PBS. Add 0.8 ml of Buffer PC-A.

2. Add 2.5 ml of Buffer PC-B pre-chilled at 4?. Pipette to mix well, incubate at room temperature for 5 min and then shake vigorously.

* Buffer PC-B can kill virus. In order to avoid contaminating laboratory, do not shake vigorously but pipette to mix well after addition of Buffer PC-A and Buffer PC-B. Stand for 5 min to kill virus, and then shake vigorously to mix well.

3. Centrifuge at =5000×g for 5 min at 4? to precipitate virus and cells on the interface.

4. Add 2 ml of Buffer VP and mix well, centrifuge at =5000 g for 3 min at 4? to pellet virus and cells on the bottom of tube.

5. Discard the supernatant. Add 5 ml of 50% ethanol, and rotate the tube to wash off the solution on the wall. Centrifuge at =5000 g for 3 min at 4?.

6. Discard the supernatant, short spin and remove all solution.

* The virus pellet should be used for purification of viral RNA immediately, or store at -20?.
 

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