CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5′ ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism – single crossing CPA – and provide details on alternative mechanisms.

Cross-priming amplification (CPA) technology for tuberculosis diagnosis from sputum specimens was evaluated. The sensitivity of CPA from smear-and liquid culture-positive specimens was 96.9%, and that from smear-negative and liquid culture-positive specimens was 87.5%. The specificity of CPA in culture-negative specimens was 98.8%.The diagnosis of tuberculosis (TB) is based on phenotypic and genotypic methods. Light microscopy for the detection of acid-fast bacilli (AFB) in sputum smears is a rapid and specific tool commonly used all over the world, but it only detects 30% to 40% of TB patients (10). While the sensitivity of Löwen-stein-Jensen (L-J) solid or liquid culture methods is higher than that of AFB microscopy, culture-based methods require several weeks of incubation time. Several genotypic assays, mostly based on nucleic acid amplification tests, have been developed for rapid TB diagnosis, including the GenProbe amplified Mycobacterium tuberculosis direct test, Roche Amplicor MTB test, Cobas Amplicor test, Abbott LCx test, and the BD-ProbeTec (strand displacement amplification) test (1,2,5,8,12). However, the cost of equipment needed for these methods is a barrier to their widespread use, especially in developing countries. A sensitive, accurate, rapid, and affordable diagnostic tool that will work in resource-limited settings is urgently needed.Cross-priming amplification (CPA) technology is a recent invention from an isothermal DNA amplification system by Ustar Biotechnologies Co., Ltd. Using multiple cross-linked primers (six to eight primers), a DNA target sequence can be amplified at a constant temperature (Fig. 1). The detection of amplified products is performed on a lateral flow strip housed in an enclosed, sealed plastic device to prevent the leakage of amplicons (4, 7). In this study, we evaluated the CPA isothermal amplification and detection kit (Ustar Biotech, Hangzhou, China) to determine whether it could accurately detect M. tuberculosis in clinical sputum specimens from a variety of different types of patients. Sputum specimens from 180 persons who were suspected of having TB, based on their clinical symptoms and presentation, were obtained from three hospitals in Shanghai. Sputum specimens were also collected from 98 non-TB patients (with lung cancer, lung edema, and other pulmonary illnesses) presenting at the same hospitals. For comparison with the CPA assay results, all of the clinical specimens in our study were also processed for AFB smear microscopy, L-J solid medium, and BacT/Alert 3D liquid culture following standard protocols (11). Bacterial genomic DNA was extracted by boiling, as previously described (9). The CPA method was performed according to the manufacturer's instructions, using primers targeting the gyrB gene of M. tuberculosis ( Fig. 2A). The result of each CPA assay was determined by observing the presence (positive result) or absence (negative result) of visible bands on the test strips (Fig. 2B).The detection rates of mycobacteria in the 180 sputum specimens used ...

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high falsenegative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity. (J Mol

A virus known as severe fever with thrombocytopenia syndrome virus (SFTSV) was recently identified as the etiological agent of severe fever with thrombocytopenia syndrome (SFTS) in China. Reliable laboratory detection and identification of this virus are likely to become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method which incorporates reverse transcription-cross-priming amplification (RT-CPA) coupled with a vertical flow (VF) visualization strip for rapid detection of SFTSV. The RT-CPA-VF assay targets a conserved region of the M segment of the SFTSV genome and has a limit of detection of 100 copies per reaction, with no cross-reaction with other vector-borne bunyaviruses and bacterial pathogens. The performance of the RT-CPA-VF assay was determined with 175 human plasma specimens collected from 89 clinically suspected SFTS patients and 86 healthy donors. The sensitivity and specificity of the assay were 94.1% and 100.0%, respectively, compared with a combination of virus culture and real-time RT-PCR. The entire procedure, from specimen processing to result reporting, can be completed within 2 h. The simplicity and nearly instrument-free platform of the RT-CPA-VF assay make it practical for point-of-care testing.

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