RT-qPCR Testing of SARS-CoV-2: A Primer.

Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability. This has resulted in widespread misrepresentation of the problems faced using this test during the current COVID-19 epidemic. This primer provides simple, straightforward and impartial information about RT-qPCR.

[Quantitative Detection of Akkermansia muciniphila in Fecal Samples by Real-time PCR].

OBJECTIVE: To develop a real-time fluorescence PCR assay for quantitative detection of Akkermansia muciniphila (A. muciniphila) in fecal samples,providing technical support for quantitative detection and deep research of A.muciniphila. METHODS: The quantitative detection method for A.muciniphila were established by using SYBR Green Ⅰ; The standard curve was made using A.muciniphila standards; The sensitivity,specificity,anti-interference and repeatability of this assay were analyzed; 30 human fecal samples were detected by the established method. RESULTS: The standard curve showed a good linear relationship with R2=0.999; The sensitivity of this assay reached to 1.0×102CFU/mL; This method could amplify A.muciniphila specifically,not affected by other intestinal bacteria; Repeatability of intra-group and inter-group were good with the coefficient of variation (CV)of Ct value lower than 2%; The positive rate of A.muciniphila in 30 human fecal samples was 73%,while its Ct value ranged from 17.40 to 31.77. CONCLUSION: These results indicated that the established real-time PCR method for A. muciniphila' quantitative detection was simple,rapid and economical,good in sensitivity,specificity,anti-interference,repeatability and suitable for the detection of A.muciniphila in faeces.

[A real-time fluorescence quantitative PCR method for source identification of Crocus sativus and Carthamus tinctorius].

The specific PCR primer was designed base on ITS2 sequence in GenBank, and we developed a SYBRGreen real-time fluorescence quantitative PCR system for identification of Crocus sativus and Carthamus tinctorius source. Compared with Chinese herbal medicine DNA barcode technique, this method showed characteristics of shorter time, higher specificity and sensitivity. Using this method to detect 15 samples, 4 were C. sativus, 8 were C. tinctorius, and the other 3 samples were none of them. The result was in accordance with Chinese herbal medicine DNA barcode. This study lays the foundation for identification of related Chinese medical materials.

A Highly Integrated and Diminutive Fluorescence Detector for Point-of-Care Testing: Dual Negative Feedback Light-Emitting Diode (LED) Drive and Photoelectric Processing Circuits Design and Implementation.

As an important detection tool in biochemistry, fluorescence detection has wide applications. Quantitative detection can be achieved by detecting fluorescence signals excited by excitation light at a specific wavelength range. Therefore, the key to fluorescence detection is the stable control of the excitation light and the accurate acquisition of weak photoelectric signals. Moreover, to improve portability and instantaneity, devices are developing in miniaturization and integration. As the core of such devices, fluorescence detectors should also have these features. Under this circumstance, we designed a highly integrated and diminutive fluorescence detector and focused on its excitation light driving and photoelectric signal processing. A current-light dual negative feedback light-emitting diode (LED) driving circuit was proposed to obtain constant current and luminance. In addition, a silicon photodiode (PD) was used to receive and convert the fluorescence signal to an electric signal. Then, amplifying, filtering, and analog-to-digital (A/D) converting were applied to make the detection of weak fluorescence signals possible. The test results showed that the designed circuit has wonderful performance, and the detector shows good linearity (R2 = 0.9967) and sensitivity (LOD = 0.077 nM) in the detection of fluorescein sodium solution. Finally, a real-time fluorescence polymerase chain reaction (real-time PCR) of Legionella pneumophila was carried out on a homemade platform equipped with this detector, indicating that the detector met the requirements of real-time PCR detection.

Effects of Different Temperature and Time Durations of Virus Inactivation on Results of Real-time Fluorescence PCR Testing of COVID-19 Viruses.

The novel Coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan, Hubei province of China in January 2020. This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2. Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13, 2020 and throat swabs were taken. The swabs were stored at room temperature (20-25°C), then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses (56°C for 30, 45, 60 min; 65, 70, 80°C for 10, 15, 20 min). Control aliquots were stored at room temperature for 60 min. Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2. Regardless of inactivation temperature and time, 7 of 12 cases (58.3%) tested were positive for SARS-CoV-2 by PCR, and cycle threshold values were similar. These results suggest that virus inactivation parameters exert minimal influence on PCR test results. Inactivation at 65°C for 10 min may be sufficient to ensure safe, reliable testing.

Analysis of digestion of rice planthopper by Pardosa pseudoannulata based on CO-I gene.

In order to systematically study the predatory behavior and digestion regularity of spiders, real-time fluorescence quantification PCR technique was used to detect the number of CO-I genes in Pardosa pseudoannulata after it preyed on rice planthoppers in different temperatures within different periods. At 28 °C, 0, 1, 2, 4, 8, 16, and 24 h after P. pseudoannulata preyed on rich planthopper, DNA was extracted from cephalothorax and abdomen of P. pseudoannulata. Routine PCR and real-time fluorescence PCR techniques were employed for CO-I gene amplification. The results show that: The prey liquid was temporarily stored in the sucking stomach of the spider head within 2 h after prey, and gradually transferred to the midgut of the abdomen with the prolongation of time. After 4 h, CO-I gene residues of rice planthopper in the cephalothorax gradually decreased. The CO-I gene of rice planthopper was basically transferred to the abdomen after 16 h. During 0-1 h, food contained in abdominal midgut and other digestive organs was very small, CO-I gene detection was not obvious. Over time, food entered into the midgut from the sucking stomach for digestion. During 2-4 h, CO-I gene amount increased, at 2-4 h, detected CO-I gene residue reached the peak; but rapidly declined after 8, 16, and 24 h, even it is still detectable. The results at different temperatures reveal that: As the temperature increased from 26 °C to 32 °C, CO-I gene residues of rich planthopper in cephalothorax and abdomen of P. pseudoannulata gradually decreased, which indicated that the digestion rate increased with the increase of temperature with some range. However, when the temperature continued to increase to 34 °C, the digestion rate decreased.