Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

First TaqMan Assay to Identify and Quantify the Cylindrospermopsin-Producing Cyanobacterium <i>Aphanizomenon ovalisporum</i>in Water

Cylindrospermopsin (CYN) is an alkaloid that causes hepatotoxicity, neurotoxicity and general cytotoxicity in vertebrates. It is currently gaining widespread attention after its reported appearance in water bodies around the world. A. ovalisporum is capable of CYN-production and can form toxic blooms when favorable environmental conditions are available. We have developed for the first time a two-step qPCR assay using Taqman probes to detect and quantify potential CYN-producing A. ovalisporum in water samples. The assay was sensitive enough to discriminate between CYN-producing and non-CYN-producing A. ovalisporum in a mixed background, and discriminate between A. ovalisporum and other nostocales as C. raciborskii and A. bergii. The detection limit of the assay falls in the log linear range of 102 and 105 gene copies per reaction and is thus within the sensitivity range of previously published assays for the detection of other toxic cyanobacteria species. Our assay allows for the first time to quickly assess water quality for the presence of potentially CYN-producing A. ovalisporum and can be easily used for the purposes of monitoring water bodies.

Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

Objective:To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods:BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stressinducible protein-1 with/without adjuvant.After three vaccinations,mice were challenged by Leishmania tropica promastigotes.Two months after challenge,the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods.Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium.For real-time PCR,DNA of the lymph nodes was extracted,equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers.The data of the two methods were compared by appropriate statistical methods.Results:Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice.In addition,wherever parasite load of a group was estimated high(or low)by one method,the estimated parasite load by another method was the same,although statistically significant differences were found between some groups.Conclusions:Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups.However,due to the lower errors and faster process,the real-time PCR method is preferred.

Complete genome sequence of a Rodent Torque teno virus in Hainan Island, China and establishment of a real-time PCR for detecting Rodent Torque teno virus 3

Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.

Feasibility of the Routine Clinical Use of a Multiplex Virus Polymerase Chain Reaction Assay Based on Blood Virus Detection in Hematopoietic Stem Cell-Transplanted Patients

Background: Multiplex virus assays are useful in immunocompromised hosts but still challenging in routine clinical settings in terms of their sensitivity, specificity, reproducibility, and time and cost performances. In recent years, we developed a qualitative multiplex virus PCR assay capable of the simultaneous detection of 13 virus species within 3 h. However, because of the multiple and concomitant nature of this virus assay, it should be validated for qualitative reliability. Materials and Methods: As a preclinical examination, this multiplex PCR was able to detect 1.25 × 103 copies/mL of 13 synthesized virus genomes and preserved same virus DNAs by the serial dilution method. Blood samples from 40 patients who underwent hematopoietic stem cell transplantation were then examined by multiplex PCR for 13 virus species, followed by quantitative real-time PCR for all 13 virus species as reference PCR when these patients developed symptoms suggestive of viral infection. Results: In 421 cumulative qualitative-quantitative tests, the multiplex PCR certainly detected 1.0 × 103 copies/mL of 5 viruses (CMV, JCV, BKV, HHV-6, ADV) that were frequently detected and thus reasonably analyzed. The positive and negative predictive values of multiplex PCR were 84.2% - 93.3% and 90.7% - 99.0%, respectively, and sensitivity and specificity were 59.0% - 83.3% and 97.2% - 99.2%, respectively, for these 5 viruses. Conclusion: From these performances, the multiplex PCR assay may be acceptable in a routine clinical laboratory setting.

Identified Bacteria and Virus in the Cerebrospinal Fluid of under Five Years Hospitalized Children for Clinical Meningitis at Panzi Hospital in the Eastern Part of DRC

Background: Meningitis remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo). Distinguishing children with bacterial meningitis from those with viral meningitis in the emergency department is sometimes difficult. Here we identified bacteria and virus in the cerebral spinal fluid (CSF) of children with meningitis. Material and Methods: This is a prospective, analytical study carried out in the Pediatrics department of Panzi Hospital in the South-Kivu province of DR Congo. Between April 2021 and March 2022, 150 of 251 collected CSF from children aged from 1 to 59 months hospitalised due to clinical meningitis at Panzi referral university hospital, Bukavu, Eastern DR Congo were sent to the Lancet laboratory for bacteria identification by a multiplex real-time PCR assay for detection of the most different viruses and bacterial species causing meningitis. Result: The used multiplex real-time PCR assay allowed us to identify germs in 24.7% of cases (37/150). We isolated bacteria in 25/37 (67.5%) cases, and viruses in 9/37 (24.3%) while virus and bacteria co-infection was detected in 3/37 (8.1%). The most frequently identified bacteria were Streptococcus pneumoniae 14/37 (37.8%) followed by Haemophilus influenzae 6/37 (16.2%). The main virus was cytomegalovirus 5/37 (3.5%). Despite the age, the most found bacterial are common in children from rural areas and unvaccinated children. Bacterial and virus co-infection were identified in 66.7% of children aged between 25 - 60 months, mainly among male children, and in all children from rural areas (100%). The overall case fatality rate was 30% and was very high among cases with co-infection CMV-Pneumococcal (66.7%), followed by Streptococcus pneumoniae (50%). Conclusion: Meningitis remains frequent among children aged from one to 59 months among Bukavu Infants. We noticed that, Children with co-infection with bacteria and viruses might need higher attention when having meningitis symptoms, as this could lead to fatal outcomes. The introduction of molecular techniques, such as multiplex real-time PCR, has the potential to improve diagnosis and patient outcomes.

右美托咪定对单肺通气患者外周血炎症反应相关基因表达的影响

目的基于PCR Assay芯片技术,分析右美托咪定对单肺通气患者炎症相关基因表达的变化并推论其可能的作用机制。方法选取本院胸科2016年1月~2016年12月的60个单肺通气病例,随机分为右美托咪定治疗组与常规治疗组,各30例。右美托咪定干预前与干预后1,3天所有患者静脉采血立即提取RNA做PCR Assay基因芯片检测。结果右美托咪定干预后炎症相关基因CCL11、CCL19、CCL2、IL17A、IL1a、IL1b、IL2的Fold绝对值趋向于干预前水平,而常规治疗组治疗前后,绝大多数Fold值变化并不大。结论右美托咪定干预使用可调节单肺通气患者炎症反应相关基因的表达,使围手术期紊乱的炎症反应趋于正常,从而起到对单肺通气患者的保护作用。

Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord iniury has a good dinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Daw- ley rats was clamped for 60 seconds. Dazhui （GV14） and Mingmen （GV4） acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expres- sion of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem ceils.

Effects of neuregulin-1 on autonomic nervous system remodeling post-myocardial infarction in a rat model

Sympathetic nerve and vagus nerve remodeling play an important part in cardiac function post-myocardial infarction （MI）. Increasing evidence indicates that neuregulin-1 （NRG-1） improves cardiac function following heart failure. Since its impact on cardiac function and neural remodeling post-MI is poorly understood, we aimed to investigate the role of NRG-1 in autonomic nervous system remodeling post-MI. Forty-five Sprague-Dawley rats were equally randomized into three groups： sham （with the left anterior descending coronary artery exposed but without ligation）, MI （left anterior descending coronary artery ligation）, and MI plus NRG-1 （left anterior descending coronary artery ligation followed by intraperitoneal injection of NRG-1 （10 lag/kg, once daily for 7 days））. At 4 weeks after MI, echocardi- ography was used to detect the rat cardiac function by measuring the left ventricular end-systolic inner diameter, left ventricular diastolic diameter, left ventricular end-systolic volume, left ventricular end-diastolic volume, left ventricular ejection fraction, and left ventricular fractional shortening, mRNA and protein expression levels of tyrosine hydroxylase, growth associated protein-43 （neuronal specific pro- tein）, nerve growth factor, choline acetyltransferase （vagus nerve marker）, and vesicular acetylcholine transporter （cardiac vagal nerve fiber marker） in ischemic myocardia were detected by real-time PCR and western blot assay to assess autonomous nervous remodeling. After MI, the rat cardiac function deteriorated significantly, and it was significantly improved after NRG-1 injection. Compared with the MI group, mRNA and protein levels of tyrosine hydroxylase and growth associated protein-43, as well as choline acetyltransferase mRNA level significantly decreased in the MI plus NRG-1 group, while mRNA and protein levels of nerve growth factor and vesicular acetylcholine transporters, as well as choline acetyltransferase protein level slightly decreased. Our results indicate that NRG- 1 can improve cardiac function and regulate sympathetic and vagus nerve remodeling post-MI, thus reaching a new balance of the autonomic nervous system to protect the heart from injury.

Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum

Objective Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain. Methods Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer. Results Arginine production assays showed a 69.9% reduction in arginine from 9.01±0.22 mg/mL in C. crenatum MT to 2.71±0.13 mg/mL （P〈0.05） in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein. Conclusion The arginine biosynthetic genes in C crenatum are clearly controlled by the regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in production. negative arginine production.

Developing a one-step triplet-repeat primed PCR assay for diagnosingmyotonic dystrophy

Myotonic dystrophy type 1 (DM1),or Steiner’s disease,is an autosomal dominant disorder caused by the expansion of unstable trinucleotide repeats (CTG) in the 3’ untranslated region of the myotonic dystrophy protein kinase gene (DMPK) (Brook et al.,1992;Mahadevan et al.,1992).The number of CTG repeats observed in normal individuals is in a range of 5-34,while the individuals with 35-49 CTG repeats are usually asymptomatic but at risk of

Identification of various cell culture models for the study of Zika virus

AIM To identify cell culture models supportive for Zika virus(ZIKV) replication.METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis.RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293 T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5 Y released 100 times less infectious viral particles than Vero-, A549-or 293 T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines.CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research.

A quantitative RT-PCR assay for rapid detection of Eurasianlineage H10 subtype influenza A virus

Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 11NA subtypes have been reported(Tong et al.,2012).

A multiplex microsatellite PCR method for evaluating genetic diversity in grass carp(Ctenopharyngodon idellus)

Grass carp is one of the most important cultured fishes all over the world.The genetic diversity of grass carp plays an important role whatever in selective breeding progress or ecological conservation purposes.However,some genetic diversity researches were not accuracy and cannot be compared with each other due to different molecular markers,sample size and detection methods.In this study,we constructed five multiplex PCR assays contained 20 microsatellites with highly polymorphism and heterozygosity for evaluating genetic information of grass carp.We used nine cultured populations consisting of 507 individuals to detect stability of the five multiplex PCR assays.The results showed that the number of alleles(Na),effective number of alleles(Ne),observed heterozygosity(Ho),expected heterozygosity(He)and polymorphism information content(PIC)of the 20 microsatellite loci were relative high compared with the genetic diversity parameters of microsatellite loci developed by other researchers.Six loci were significantly deviated from Hardy-Weinberg equilibrium(P<0.01).And with exception of the Shaoguan,Indian and Nepal cultured population,all other cultured populations showed very high genetic diversity.Through the test of grass carp populations,we developed an effective and accurate multiplex SSR-PCR assays that can be as statistical powerful tool for detecting genetic information of grass carp.

VALUE OF POLYMERASE CHAIN REACTION ASSAY IN DIAGNOSIS OF INTESTINAL TUBERCULOSIS AND DIFFERENTIATION FROM CROHN'S DISEASE

It is difficult to make a precise diagnosis of intestinal tuberculosis and to differentiate it from Crohn's disease. For evaluating Polymerase Chain Reaction (PCR) assay in these two aspects, 36 specimens of intestinal tuberculosis from surgical resections and endoscopic biopsies and 26 Crohn's disease samples were subjected to PCR assay. 21 specimens of normal colon tissue surrounding cancer were used as the control. Oligonucleotides derived from the IS 6110 sequence, which is repeated in M. tuberculosis chromosome and highly specific for the M. tuberculosis complex, were used as a primer. The amplified PCR products were detected by examination of ethidium-bromide-stained polyacrylamide gels. The specificity of PCR products was confirmed by digestion with Sal 1 restrictive endonuclease and southern blot hybridization using digoxigenin-labeled probe. The results showed that the M. tuberculosis DNA was identified in 27 / 36 intestinal tuberculosis, but none of 26 Crohn's disease. Acid fast bacilli were only found in 16 / 36 intestinal tuberculosis. In conclusion, as a rapid, sensitive, and specific pathogenic method in diagnosis of intestinal tuberculosis, PCR assay has been developed in this study, and is considered valuable in the differentiation between intestinal tuberculosis and Crohn's disease.

A Multicenter Study of Viral Aetiology of Community-Acquired Pneumonia in Hospitalized Children in Chinese Mainland

Community-acquired pneumonia(CAP) is one of the leading causes of morbidity and mortality in children worldwide.In this study,we aimed to describe the aetiology of viral infection of pediatric CAP in Chinese mainland.During November2014 to June 2016,the prospective study was conducted in 13 hospitals.The hospitalized children under 18 years old who met the criteria for CAP were enrolled.The throat swabs or nasopharyngeal aspirates(NPAs) were collected which were then screened 18 respiratory viruses using multiplex PCR assay.Viral pathogens were present in 56.6%(1539/2721) of the enrolled cases,with the detection rate of single virus in 39.8% of the cases and multiple viruses in 16.8% of the cases.The most frequently detected virus was respiratory syncytial virus(RSV)(15.2%,414/2721).The highest detection rate of virus was in <6-month-age group(70.7%,292/413).RSV,human metapneumovirus(HMPV),human parainfluenza viruses(HPIVs) and influenza B virus(Flu B) showed the similar prevalence patterns both in north and south China,but HPIVs,Flu A,human bocavirus(HBoV),human adenovirus(HAdV) and human coronaviruses(HCoVs) showed the distinct circulating patterns in north and south China.Human enterovirus/human rhinovirus(HEV/HRV)(27.6%,27/98),HBoV(18.4%,18/98),RSV(16.3%,16/98) and HMPV(14.3%,14/98) were the most commonly detected viruses in severe pneumonia cases with single virus infection.In conclusion,viral pathogens are frequently detected in pediatric CAP cases and may therefore play a vital role in the aetiology of CAP.RSV was the most important virus in hospitalized children with CAP in Chinese mainland.

Knowing your enemies： Integrating molecular and ecological methods to assess the impact of arthropod predators on crop pests

The importance of natural enemies as the foundation of integrated pest management （IPM） is widely accepted, but few studies conduct the manipulative field experiments necessary to directly quantify their impact on pest populations in this context. This is particularly true for predators. Studying arthropod predator-prey interactions is inherently difficult： prey items are often completely consumed, individual predator-prey interactions are ephemeral （rendering their detection difficult） and the typically fluid or soft-bodied meals cannot be easily identified visually within predator guts. Serological techniques have long been used in arthropod predator gut-contents analysis, and current enzyme linked immunosorbent assays （ELISA） are highly specific and sensitive. Recently, poly- merase chain reaction （PCR） methods for gut-contents analysis have developed rapidly and they now dominate the diagnostic methods used for gut-contents analysis in field-based research. This work has identified trophic linkages within food webs, determined predator diet breadth and preference, demonstrated the importance of cannibalism and intraguild predation within and between certain taxa, and confirmed the benefits （predator persis- tence） and potential disadvantages （reduced feeding on pest species） of the availability of alternative nonpest prey. Despite considerable efforts to calibrate gut-contents assays, these methods remain qualitative. Available techniques for predator gut-contents analysis can provide rapid, accurate, cost-effective identification of predation events. As such, they perfectly compliment the ecological methods developed to directly assess predator im- pacts on prey populations but which are imperfect at identifying the key predators. These diagnostic methods for gut-contents analysis are underexploited in agricultural research and they are almost never applied in unison with the critical field experiments to measure predator impact. This paper stresses the need for a combined approach and suggests a framework that would make this possible, so that appropriate natural enemies can be targeted in conservation biological control.